Redirected, genetically-engineered t regulatory cells and their use in suppression of autoimmune and inflammatory disease

ABSTRACT

A redirected Treg cell is endowed with specificity toward a selected target antigen or ligand. The cell contains a chimeric receptor polypeptide that is expressed in a single, continuous chain, with an extracellular recognition region displayed on the surface of the cell, a transmembrane region and an intracellular signaling region. The extracellular recognition region is specific for the selected target antigen or ligand. The intracellular signaling region includes a combination of T-cell signaling polypeptide moieties, which combination, upon binding of the extracellular recognition region to the selected target antigen or ligand, triggers activation of the redirected Treg cells to cause suppression of T-cell mediated immunity. Such redirected Treg cells may be used to suppress undesired activity of T effector cells thereby mediating an immune or inflammatory response. They are particularly useful in treating T effector cell-mediated diseases, such as inflammatory bowel disease, transplant rejection and GVH disease.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention in the field of immunology and medicine relates to geneticmodification of T regulatory cells with chimeric receptors withantibody-type specificity, and the use of such cells to suppress theaction of T effector cells and treat any of a number of diseases andconditions in which such suppression is beneficial, primarily autoimmuneand inflammatory diseases such as inflammatory bowel diseases (IBD),organ-specific autoimmune diseases, allograft rejection and Graft-vs.Host disease.

2. Description of the Background Art

Regulatory T-Cells (Tregs)

One line of research that led to discovery of Treg cells was theobservation that thymectomy of mice of certain susceptible strains onpostnatal day 3 results in a spectrum of organ-specific autoimmuneeffects, which were preventable by “reconstitution” of these animalsearly in life with normal adult lymphocytes (Asano M et al., J Exp Med1996; 184:387-96). The effectors and suppressors of autoimmunity in thismodel of multi-organ autoimmunity were CD4+T-cells. It was subsequentlyshown that the regulatory CD4+Tregs that prevent disease coexpressedCD25. CD4+CD25+ Tregs represent 5-10% of total peripheral CD4+ cells inmice and 3-6% of total peripheral blood CD4+T-cells in humans (JonuleitH et al., J Exp Med 2001; 193:1285-94).

Over the past decade, CD4+CD25+ Tregs have been studied for theirfunction in autoimmune disease. CD4+CD25+ Tregs suppressed diseaseinduced by cloned autoantigen-specific T effector cells (Sufi-Payer, Eet al., J. Immunol., 1998; 160:1212-18). The CD4+CD25+ Treg cellsappeared to be members of a unique lineage of regulatory T-cells. Theseauthors noted that, although the target antigen(s) and mechanism ofaction of the CD4+CD25+T-cells remained to be determined, they likelyplayed an important role in modulating other autoimmune diseases thatare mediated by activation of self-reactive T-cells. Tregs preventorgan-specific autoimmune diseases including autoimmune thyroiditis,autoimmune gastritis, insulitis and arthritis.

More recently, it was discovered that Tregs express a transcriptionfactor known as Foxp3 intracellularly. The absence of the transcriptionfactor called Scurfin (also forkhead box P3) and encoded by the geneFoxp3 was known to cause a rapidly fatal lymphoproliferative disease,similar to that seen in mice lacking cytolytic T lymphocyte-associatedantigen 4 (CTLA-4). Khattri R et al. (Nat Immunol. 2003; 4:337-42)showed that Foxp3 was highly expressed by Treg cells and was associatedwith their activity and phenotype. Foxp3-deficient mice lacked Tregcells whereas mice that overexpressed Foxp3 possess more Treg cells.Tregs constitutively express the transcription factor Foxp3 and theinhibitory costimulatory molecule CTLA-4 (Chen W et al., J Exp Med 1998;188:1849-57.

Foxp3 is believed to act through negative transcriptional regulation ofcytokine genes, including IL2, IL4 and IFN-γ (Kasprowicz D J et al., JImmunol. 2003; 171:1216-23, 2003), though many aspects of Foxp3 activityand regulation of its expression remain obscure.

Loser K et al., Gene Ther 2005; 12:1294-304, generated Tregs in vitro byinfecting naïve CD4+CD25−T-cells with a retrovirus encoding Foxp3.Foxp3-infected T-cells were similar to naturally occurring Treg cells asevidenced by surface marker expression and function. These authorsinvestigated the effects of Foxp3-infected T-cells on contacthypersensitivity responses mediated by T effector cells by injectinginto sensitized mice Foxp3- or control virus-infected T-cells. Onlyinjection of Foxp3-infected T-cells significantly inhibited CHS comparedto controls, indicating that Foxp3-infected T-cells are suppressive invivo. The authors then used Foxp3-infected T-cells to treatautoimmune-prone CD40L transgenic (Tg) mice, which develop a severesystemic autoimmune disease including autoreactive T-cells andautoantibodies. Injection of Foxp3-infected T-cells into these miceinhibited the ongoing development of autoimmune dermatitis andactivation of cytotoxic CD8+T-cells. This treatment also reduced serumconcentrations of antinuclear antibodies, which was paralleled withreduced renal immunoglobulin depositions and increased kidney function.The authors concluded that newly in vitro-generated regulatory T-cellscan be used to treat inflammatory and ongoing autoimmune disorderssuccessfully.

Suri-Payer E and Fritzsching B (Springer Semin Immunopathol. 2006;28:3-16) recently summarized evidence for a role for Treg in suppressionof innate and adaptive immune responses in experimental models ofautoimmunity including arthritis, colitis, diabetes, autoimmuneencephalomyelitis, lupus, gastritis, oophoritis, prostatitis, andthyroiditis. A common observation from such studies is that Tregs areactivated in an antigen-specific manner, but exert their suppressivefunction in an antigen-independent manner, mainly by producing andsecreting suppressive cytokines such as IL-10 and TGF-β. Tregs cansuppress “conventional” T-cells in vitro by direct cell contact. It isappreciated, however, that down-regulation of antigen-presenting cell(APC) function, such as that of dendritic cells, and attenuation ofsecretion of inhibitory cytokines such as IL-10 and TGF-β might beimportant for Treg function in vivo. The final outcome of autoimmunityvs. tolerance depends on the balance between stimulatory signals to Teffector cells and inhibitory signals from Treg. Whereas earlier studiesanalyzed the capacity of Tregs to prevent onset of autoimmune disease,more recent reports indicate successful treatment of ongoing disease.

In vivo, adoptive transfer of Tregs achieved the following effects:

(1) suppressed development of autoimmunity;

(2) suppressed acute rejection of transplanted solid organs; and

(3) suppressed anti-tumor immunity,

A review of Sakaguchi S et al., Immunol Rev. 2006; 212:8-27, noted thatnaturally arising CD25+CD4+ Treg cells play key roles in the maintenanceof immunologic self-tolerance and negative control of a variety ofphysiological and pathological immune responses. Most of these cells areproduced by the normal thymus as a functionally mature T cellsubpopulation. Natural Tregs specifically express Foxp3, a transcriptionfactor that plays a critical role in their development and function.Complete depletion of Foxp3-expressing natural Tregs (whether they areCD25+ or CD25−) activated even weak or rare self-reactive T effectorcell clones, inducing severe and widespread autoimmune/inflammatorydiseases. Natural Tregs are highly dependent on exogenously providedinterleukin (IL)-2 for their survival in the periphery. In addition toFoxp3 and IL-2/IL-2 receptor, a deficiency or functional alteration ofother molecules (expressed by T-cells or non-T-cells), may affect thedevelopment or function of Tregs, self-reactive T effector cells, orboth, and consequently tip the balance between the two populations inthe periphery toward autoimmunity. Thus, Tregs suppress the activity ofT effector cells that are a major cause of antigen-specific autoimmuneinflammatory disorders. Tregs induce anergy and promote suppression by aprocess that involves both cell-cell contact, and probably moreimportantly, by their secretion of TGF-β and IL-10.

Sakaguchi et al., supra, stated that elucidation of the molecular andcellular basis of Treg-mediated active maintenance of self-tolerancewill facilitate (1) our understanding of the pathogenesis of autoimmunedisease and (2) development of novel methods of autoimmune diseaseprevention and treatment.

The present invention is directed to one such novel approach to preventor treat autoimmune disease and related immune/inflammatory conditionsby imposing Treg-mediated control over T effector cells.

Engineering of Chimeric Receptors with Antibody Specificity into T-Cells

Efforts to confer antibody-like specificity to T lymphocytes arose as aresponse to certain basic discoveries and the failure to convert them totherapeutic success. Human tumor-associated antigens have been shown toexist as peptides associated with major histocompatibility complex (MHC)proteins. The lack of success in vaccinating tumor patients with a largevariety of tumor-associated antigen vaccines were thus frustrating(Rosenberg S A et al., Nat Med 2004; 10:909-15). Passive vaccinationwith anti-tumor antibodies (Abs), tumor infiltrating lymphocytes (TILs),or lymphokine activated killer (LAK) cells showed very limitedeffectiveness (primarily for vascular tumors). More recent successeswith the adoptive transfer of TILs into stage 1V melanoma patients((Dudley M E, et al., J Immunother 2001; 24:363-73; Dudley M E, et al.,Science 2002; 298:850-4) were still burdened by the fact that theresults were limited to a few cancers and to individuals from whom it ispossible to derive specific TILs.

To overcome such limitations in adoptive cellular immunotherapy ofcancer, and in order to develop an approach that was not restricted toindividual patients or limited to a specific form of cancer, one of thepresent inventors and his colleagues developed the “T-body” approach(Gross G et al., Proc Natl Acad Sci USA, 1989; 86:10024-8; Gross G andEshhar, Z, FASEB J 1992; 6:3370-8), See also the following patentpublications by Eshhar and colleagues, all of which are incorporated byreference in their entirety: US Pat Pub 2002-0137697, U.S. Pat. No.5,912,172, U.S. Pat. No. 5,906,936, International Patent publicationsWO00/31239, WO97/15669, WO95/14710 and WO93/19163. In these approaches,a chimeric receptor (CR), was made, for example, by fusing the variableportion of an antibody, such as an anti-tumor monoclonal antibody (mAb),to a lymphocyte intracellular triggering domain, so as to be expressedby the T-cell into which the gene has been transfected as theextracellular domain of that T-cell triggering molecule. Following theexpression of such CR genes in immune effector cells (T-cells andnatural killer (NK) cells), the resulting engineered cells (nicknamed“T-bodies”) recognized their tumor targets and efficiently killed them.Thus, the chimeric immune receptor confers redirected antigenicspecificity coupled to direct, MHC-independent triggering of cellularactivation in response to binding of pre-defined target antigen.

Originally, the heterodimeric CR configuration comprised the two T cellreceptor (TCR) α and β chains in which each pair of TCR variable domains(V_(α) and V_(β)) was replaced with a pair of V_(H) and V_(L) domainderived from a selected antibody. These two Ab-derived coding sequenceswere co-transfected into T cell lines and were found to confer antibodyspecificity (Gross et al., 1989, supra). Thus, T-cells could beactivated to effector function such as cell killing or cytotoxicactivity against an immunologically specific target in a manner that wasMHC independent (and thereby non-restricted) (Gross et al., 1992,supra).

In “second generation” CR's, the single chain configuration of the CRwas further manipulated to obtain a configuration that would be usefulparticularly for cancer or antiviral therapy. Here, a single chain Fv(scFv) of an antibody was linked to transmembrane and cytoplasmicdomains of lymphocyte triggering moieties such as the TCR/CD3complex-associated ζ chain, or the Fc receptor γ chain (Eshhar Z et al.,Proc Natl Acad Sci USA 1993; 90:720-4). This single chain configuration,which combined antibody recognition and T cell signaling in a singlecontinuous protein, was a modular structure with functional domains thatare simple to manipulate, and could be readily expressed in humanlymphocytes using retrovirus-based vectors (Eshhar Z et al., J Imm Meth2001; 248:67-76). Other such receptors designed by some of the presentinventors and colleagues, and discussed in more detail below, arereferred to as tripartite chimeric receptors (TpCR) that also include acostimulatory domain(s) (e.g. CD28, 4-1BB).

With time, it has emerged that redirection of the specificity of Teffector cells using single chain CRs has become a valid therapeuticoption for cancer. Many investigators have adapted this “T-body”approach to endow T-cells with various specificities and functions(Gross & Eshhar, supra; Willemsen R A et al., Hum Immunol 2003;64:56-68; Baxevanis C N et al., Cancer Immunol Immunother 2004;53:893-903). For a recent review, see: Eshhar, Z. “The T-Body ApproachRedirecting T Cells with Antibody Specificity,” in TherapeuticAntibodies. Handbook of Experimental Pharmacology 181, Chemajovsky &Nissim (eds.), Springer-Verlag, 2008, pp. 329-342.

In the present invention, the inventors have conceived expanded uses ofsuch CR's for the treatment of undesired immune/inflammatory conditionssuch as autoimmune diseases (with a particular initial emphasis oninflammatory bowel disease, IBD) and graft rejection.

Chronic Inflammatory Disease and Animal Models

Inflammatory conditions, particularly chronic inflammatory diseases, areof particular importance in clinical medicine. These diseases, caused byactions of the immune system, involve inappropriate or excessiveactivation of certain T-cells, expression of regulatory cytokines andchemokines, loss of immune tolerance, and the like. Examples ofautoimmune and/or chronic inflammatory diseases are multiple sclerosis,inflammatory bowel diseases (IBD), joint diseases such as rheumatoidarthritis, and systemic lupus erythematosus. Some of these diseases arerather organ/tissue-specific as follows: intestine (Crohn's Disease),skin (psoriasis), myelinated nerves (multiple sclerosis or MS),pancreatic islet or β cells (insulin dependent diabetes mellitus (IDDM)or Type I Diabetes), salivary glands (Sjogren's disease), skeletalmuscle (myasthenia gravis), the thyroid (Hashimoto's thyroiditis;Graves' Disease), the anterior chamber of the eye (uveitis), jointtissue (rheumatoid arthritis), and various cardiovascular diseases.

Inflammatory bowel disease (IBD) is a collective term used to describetwo intestinal disorders whose etiology is not completely understood:Crohn's disease and ulcerative colitis. IBD occurs worldwide andafflicts several million people (0.3% of people in Western countries),and its incidence is on the rise (Tsironi E, et al., Am J Gastroenterol.2004; 99:1749-55). The course and prognosis of IBD varies widely. Onsetof IBD is predominant in young adulthood and presents typically withdiarrhea, abdominal pain, and fever; anemia and weight loss are alsocommon signs. Between 10% and 15% of people with IBD require surgeryover a ten year period. Patients with IBD are also at increased risk forthe development of intestinal cancer. These diseases are accompanied bya high frequency of psychological symptoms, including anxiety anddepression.

Although the pathogenesis of this common T cell-mediated disorderremains uncertain, it is believed to result from loss of tolerance inthe intestinal immune system due to the presence of constant antigenicstimulation provided by the very large numbers of resident bacteria(Podolsky D K, N Engl J Med 2002; 347:417-29). Unfortunately, newtherapies for IBD are few, and both diagnosis and treatment have beenhampered by a lack of detailed knowledge of the etiology. A combinationof genetic factors, exogenous triggers and endogenous microflora cancontribute to the immune-mediated damage of intestinal mucosa. Bacteriahave been implicated in initiation and progression of Crohn's Diseasesince intestinal inflammation frequently responds to antibiotics. Commonintestinal colonists and novel pathogens have been implicated, eitherbecause of direct detection or disease-associated anti-microbial immuneresponses. In many genetically susceptible animal models of chroniccolitis, luminal microorganisms appear to be a necessary cofactor fordisease.

The initiating step in autoimmune disease pathology is often obscure inhumans where the diseases are largely sporadic, and symptoms may appearyears after the first pathogenic T cell is activated. It has thereforebeen difficult to design effective therapies to block induction ofdisease. In contrast, there are common features in many of the laterstages of these diseases. Inflammation at the disease site/target organis typically present, caused by the release of inflammatory, also termed“proinflammatory,” cytokines (e.g. TNF-α and interferons) by T-cells andby other cells that contribute to the activation steps and effectorpathways of immune/inflammatory processes. These cells include (amongothers) macrophages, dendritic cells and their precursors, B lymphocytesand plasma cells and NK cells (including NKT-cells). These reactionsoften involve destruction of “target” cells and tissue damage.

Studies using murine models of experimental chronic inflammation arehelping to define nature of the immunological dysregulation thatinitiates inflammation and leads to destruction of specific end organsas well as for testing therapies. See, for example, Mombaerts et al.Cell, 1993; 75:274-82; Tarrant et al., 2998; J Immunol, 161:122-7;Powrie et al., Immunity, 1994; 1:553-62; Hong et al., J Immunol, 1999;162:7480-91; Horak, Clin Immunol Immunopathol, 1995, 76(3 Pt2):5172-173; Ehrhardt et al. J Immunol, 1997; 158:566-73; Davidson etal., J Immunol, 1998; 161:3143-9; Kuhn et al. Cell, 1993. 75:263-74;Neurath et al., J Exp Med, 1995. 182:1281-90. W. Strober, 2002; Annu.Rev. Immunol. 20:495-54 reviews mucosal models of inflammation, and isincorporated by reference in its entirety. One hallmark of the better ofthese models is that the histopathology and pathophysiology resemblesthat of the parallel human conditions, further enhancing the models'utility in testing novel treatment strategies. In the case of IBD thisdevelopment has not been uniform. Most emphasis has been placed onmodulation of immune mechanisms (Blumberg R S et al., Curr Opin Immunol.1999; 11:648-56; Strober et al., supra) and recently of the entericflora (Sartor R B, Curr Opin Gastroenterol. 2001; 4:324-330).

Bhan A K et al., 1999 Immunol Rev 169:195-207 reviewed studies ofcolitis in transgenic (Tg) and knockout (KO) animal models for mucosalinflammation in IBD. Genetics and the environment, particularly thenormal enteric flora, were factors in the development of mucosalinflammation, as stated above. Normal mucosal homeostasis was disruptedby cytokine imbalance, abrogation of oral tolerance, breach ofepithelial barriers, and loss of immunoregulatory cells. Some but notall immunodeficiencies, in the appropriate setting, led to colitis.CD4+T effector cells have been identified as the pathogenic lymphocytesin colitis, and can mediate inflammation by either the Th1 or the Th2pathway. The Th1 pathway dominates in most colitis models (and humanCrohn's Disease). In contrast, in the colitis observed in mice, the Tcell receptor (TCR) a chain knockout mice (TCRα KO mice) shared manyfeatures of ulcerative colitis including the dominance of Th2 pathway incolon inflammation. Such models are important for the development oftherapeutic strategies to treat IBD. In a later review, the same group(Mizoguchi A et al., 2003, Inflamm Bowel Dis. 9:246-259) noted thatexaggerated immune responses to normal enteric microflora are involvedin the initiation and perpetuation of chronic intestinal inflammation. Amajor pathway involves development of “acquired” immune responses by theinteractions of CD4+TCRαβ+T-cells with antigen-presenting cells (APC),particularly dendritic cells. CD4+CD25+ Treg cells attenuated activatedT cell responses.

The progression from the acute to the chronic phase of IBD has not beenwell characterized in animal models and cannot be easily evaluated inpatients. Spencer D M et al. Gastroenterol. 2002; 122:94-105 reportedchanges in the mucosal immune response over time in experimentalcolitis. Severity of colitis, body mass, stool consistency and bloodcontent, serum amyloid A, and tissue histology were examined in micedeficient in interleukin-10 (IL-10) over 35 weeks. The correspondingproduction of IL-12, IL-18, IFNγ, TNFα, IL-4, and IL-13 by laminapropria mononuclear cells in the inflamed intestine was measured.Administration of a neutralizing anti-IL-12 monoclonal antibody (mAb) atdistinct times during disease progression permitted evaluation of thetherapeutic potential of this agent. Lamina propria mononuclear cellsfrom mice with early disease synthesized progressively more IL-12 andIFNγ, whereas production of both cytokines declined dramatically andreturned to pre-disease levels in the late phase. Consistent with thispattern, the neutralizing anti-IL-12 reversed early, but not late,disease. In contrast, IL-4 and IL-13 production increased progressivelyfrom pre- to early to late disease. It was concluded that colitisdeveloping in IL-10-deficient mice evolves into two distinct phases.IL-12 plays a pivotal role in early colitis, whereas other immunemechanisms, presumably mediated by IL-4 and IL-13, predominate in latedisease to sustain chronic inflammation.

IL-10 and Chronic Inflammatory Disease

It has been known for some years that IL-10 affects the growth anddifferentiation of many hemopoietic cell types in vitro and is aparticularly potent suppressor of macrophage and T cell functions. Theseobservation were based in part from use of IL-10-deficient (knockout,KO) mutant mice by gene targeting (Kuhn R et al., Cell 1993; 75:263-74).In these mice, lymphocyte development and antibody responses are normal,but most animals are growth retarded, anemic and suffer from chronicenterocolitis. Alterations in the intestine include extensive mucosalhyperplasia, inflammation, and aberrant epithelial expression of majorhistocompatibility complex (MHC) class II molecules. In contrast, IL-10KO mutants kept under specific pathogen-free conditions, develop onlylocalized inflammation (limited to the proximal colon). It was concludedthat (1) bowel inflammation in these mutants originated fromuncontrolled immune responses stimulated by enteric antigens and (2)IL-10 is an essential (negative) regulator in the intestinal tract.

In a study validating this IL-10 KO mouse model of colitis, T. Scheinin,T et al. (Clin Exp Immunol 2003; 133:38-43) emphasized that a valuableanimal model must respond to existing therapy in a way that resemblesthe response of human disease. Since refractory Crohn's Diseaseresponded well to anti-TNFα antibody therapy, the investigators examinedresponses of IL-10 KO mice to anti-TNFα therapy, using a new scoringsystem similar to the Crohn's Disease “Activity Index” in humans. Stoolsamples were tested for cytokines and the findings compared withhistology. Results showed that anti-TNF antibody therapy starting at 4weeks markedly ameliorated the disease (as judged by the clinical scoreor by gut histology). A marked diminution of inflammatory cytokines instool samples was noted, adding a further accurate measure of clinicalimprovement. The authors concluded that this model is useful forevaluating other therapeutic modalities of relevance to Crohn's Disease.

Treg Cells and Inflammatory Bowel Disease

One of the commonly used animal models of IBD involves adoptive transferof CD45RB^(hi)CD4+T-cells into SCID mice, leading to the development ofmassive colon mononuclear cell infiltrates, epithelial cell hyperplasiaand ulceration (Thornton A M, et al., J Immunol 2000; 164(1):183-90).Cotransfer of large numbers of CD4+CD25+ Tregs prevented the developmentof colitis or cured established colitis, an affect that requiredsignaling through CTLA-4 (Read S, et al., J Exp Med 2000; 192:295-302;2000; Morrissey P J, et al., J Exp Med 1993; 178:237-44). Even after thedevelopment of immune-mediated colitis, adoptive transfer of 10⁶CD4+CD25+ cells caused significant improvement of intestinalinflammation (Fantini M C et al., Gut 2006; 55:671-80; Mottet C, et al.,J Immunol 2003; 170(8):3939-43; Uraushihara K, et al., J Immunol 2003;171:708-16).

In humans suffering from IBD, peripheral regulatory CD4+CD25+ cellsretain suppressive activity. However, in contrast to other intestinalinflammatory disorders, the number of these regulatory cells decreasesin peripheral blood during active inflammation and only slightlyincreases in intestinal lesions (Maul J, et al., Gastroenterology 2005;128(7):1868-78). This aberration suggests that Treg homing defects, aswell as dysregulated in situ activation contribute to the pathogenesisof IBD

There is therefore a recognized need in the art to find modalities tosuppress autoimmune/inflammatory reactions and diseases, including butnot limited to IBD, as well as to suppress rejection of organ and tissuegrafts and prevent Graft vs. Host (GVH) disease. The present inventionprovides a novel approach, that of redirecting Treg cells, as a means torecruit Tregs to sites of inflammation, and activate them to suppresssuch immune/inflammatory reactions and protect against, alleviate andeven cure such disease as IBD.

SUMMARY OF THE INVENTION

The present invention is based on the inventors' conception thatCR-mediated redirection and activation of Treg cells at sites ofinflammation results in suppression of inflammatory conditions, commonlypart of organ-specific autoimmune disease and exemplified herein asinflammation in the colon in experimental IBD. The inventors havefurther conceived of using these cells to overcome rejection ofmismatched cells and tissues by T effector cells that arise intransplant recipients or to inhibit the pathogenic action oftransplanted immunocompetent cells in the case of GVH disease.

The invention relies on the inventors' innovative T-body approach thathas thus far proven useful for immunotherapy of cancer (and is currentlyin phase I/II clinical trials). The invention provides a new approach tothe exploitation of Treg cells for amelioration of pathologic andundesired immune responses, particularly immunotherapy of autoimmune andinflammatory conditions, including various organ-predominant autoimmunediseases, and other pathologic or undesirable immune responses such asgraft rejection and graft vs. host disease.

According to this invention, Treg cells are endowed with CRs that arespecific for a selected target antigen or ligand. Such modificationcauses activation of redirected Tregs at sites of inflammation tosuppress the proinflammatory effector-type immune responses. Based onthe present inventors' (and their colleagues') results with redirectingantitumor effector lymphocytes, it is expected that Tregs, endowed withpredefined specificity, will migrate/home to and accumulate in, atargeted site, such as the inflamed colon, where they will suppressdisease-mediating T effector cells. To avoid the necessity of migrationor homing to the targeted site, the Tregs may, where possible, beadministered directly at or to such site, where they will becomeactivated and suppress disease-mediating T effector cells.

Such redirected Treg cells, also referred to as “T-bodies,” are Tregcells that have been genetically engineered to express a CR, preferablya tripartite chimeric receptor (TpCR) that is made of a single chainextracellular recognition unit, a transmembrane region, and anintracellular signaling region.

The extracellular recognition region is specific for a selected targetantigen or ligand and may preferably be a single chain antibody variable(scFv) region or another ligand that is capable of binding to the targetantigen or ligand. The extracellular recognition region preferably doesnot comprise an MHC protein extracellular domain. The redirected Tregsof the present invention are sometimes referred to herein as “T-bodies”despite the fact that the extracellular recognition region is notnecessarily an antibody domain. Thus, this term is not intended to belimited to Tregs with antibody-like specificity, but also includes Tregswith ligand-receptor-like specificity or otherwise.

A flexible spacer region may be present between the extracellularrecognition region and the transmembrane region. Such flexible spacer ispreferably an immunoglobulin (Ig)-like hinge, such as any hinge regionderived from the Ig superfamily.

The intracellular region includes a combination of T-cell signalingpolypeptide moieties, fused in tandem, which combination of moieties,upon binding of the extracellular recognition region to the selectedtarget antigen or ligand, triggers activation of the Treg cells to causesuppression of T-cell mediated immunity. The T-cell signaling moietiespreferably include one or more cytoplasmic domains of a costimulatorymolecule (e.g., CD28) and a cytoplasmic T-cell stimulatory domain, e.g.,of FcRγ or a CD3-ζ chain. The redirected Treg cells become specificallyactivated, upon binding of the extracellular recognition region of theCR to its target antigen or ligand, in a manner that is preferably (1)not restricted by, or dependent upon, the binding of the target antigenor ligand to an MHC, nor is it otherwise dependent in any way on the MHC(HL-A) haplotype of the recipient and (2) independent of engagement ofcostimulatory ligand(s) on a target cell.

A preferred target disease of this invention is an IBD such asulcerative colitis, in which the present methods, as used successfullyin an animal model, will permit Treg cells to reach bowel lesions in IBDpatients and become efficiently activated at the inflammation site. Thepresent invention results in site-specific Treg accumulation, ultimatelyresulting in CR-mediated, antigen-specific activation that results inthe production of suppressive cytokines which in turn suppress effectorautoimmune T-cells in an antigen-nonspecific manner, leading toalleviation of symptoms and thereby treating the disease.

The present therapeutic approach has several unique advantages. Incontrast to other immunotherapeutic models, it involves T-bodiesredirected with a CR, and preferably a TpCR, that combinesantibody/antigen or ligand/receptor recognition with stimulatory andcostimulatory motifs. Thus, T-bodies can be fully activated in a waythat is not restricted by the MHC and is independent of a requirementfor costimulation. The second advantage of the present invention stemsfrom the fact that, although Treg activation is antigen-dependent, thesuppressive action of these cells is antigen-, TCR-, andMHC-independent. By exploiting this property, one can construct achimeric receptor that is specific for one or more tissue-associatedantigens rather than requiring specificity for an unknown number of yetundefined autoimmune disease-specific antigens. Expression of suchchimeric receptors in Tregs redirects these cells and their activationto the appropriate target tissue (in a preferred embodiment, the colon)so that they are activated in an antigen-specific manner, where theirpotent suppressive effects take place without a need for furtherrecognition of disease-associated-antigens (the “bystander effect”). Byusing specifically-activated Tregs, many fewer cells are required totreat autoimmune inflammatory conditions; such as IBD, orallograft-associated reactions in patients than would have been possibleprior to this invention when much higher numbers of non-specific Tregswould have been needed.

The present inventors have constructed strains of transgenic (Tg) micewhose T-cells and natural killer (NK) cells express an antigen-specificTpCR. For exemplification of the invention, the inventors selected thetrinitrophenyl (TNP) hapten as the specific target of the TpCRs. TNPspecific Tregs isolated from these Tg mice suppressed TNP-specificeffector T-cells in vitro and in vivo and were able to suppresstrinitrobenzenesulfonic acid (TNBS)-induced colitis in mice. In thisembodiment, the target antigen for Treg and the pathogenic antigen—thehapten TNP—are the same. In another example, TNP-specific Tregssuppressed oxazolone-induced colitis in mice in which a low dose of TNPwas introduced into the colon together with the oxazolone challenge.However, the TNP-specific Tregs had no effect on the oxazolone-inducedcolitis in the absence of TNP introduction. This establishes the“bystander” effect of the present invention, i.e., that the targetantigen need not be the pathogenic antigen, as long as the redirectedTregs are activated in the vicinity of the pathogenic or undesiredimmune response.

One distinct advantage of the present invention is that it providescells and methods that permit antigen-specific activation andantigen-nonspecific action of Treg cells used to suppress effector Tcell responses (and treat consequent pathologies) in a way that does notrequire identity between the ligand (e.g., the antigen) recognized bythe TpCR (e.g., by its target recognition portion) and theligand/antigen that plays a pathogenic role in the disease process.Thus, the antigen that is pathogenic does not have to be recognized bythe T effector cells being suppressed, and, indeed, may be unrelated tothe disease or condition being treated. Thus, the invention exploits the“bystander” effect. As long as the Treg is in the correct vicinity whereT effector cells are located and mediating their undesired effects, theredirected Tregs of the present invention can be triggered or activatedat that location to release of suppressive cytokines (e.g., IL-10 andTGF-β), that will result in suppression of any “bystander” effectorT-cells, and by this mechanism, quell an ongoing inflammatory/autoimmuneresponse.

The unique characteristics of the Tg system used in the present examplesenables evaluation of the suppressive effect of antigen-specific Tregsin IBD both in vitro and in vivo. Different means are used to induceTregs, allowing those of skill in the art to select the optimal methodfor generating efficient numbers of antigen-specific redirected Tregsfor a desired antigen or disease/condition. According to this invention,redirected human Tregs constitute an effective cell-based therapeuticmodality for IBD or ulcerative colitis and, more broadly, for any Teffector cell-mediated disease or condition.

To overcome the scarcity of antigen-specific Tregs, the presentinvention includes methods to induce these cells using cytokines (e.g.TGF-β) or by expression of transgenes (e.g. encoding the Foxp3transcription factor) that will, together with the TpCR, allowantigen-specific Treg expansion.

According to the present invention, human Treg cells, derived fromeither the subject with the autoimmune/inflammatory disease or conditionto be treated, or from an HLA-matched healthy donor (or a universal cellthat is not recognized by the recipient's immune system), are endowedwith antigen/ligand-specificity, by transduction with theantigen/ligand-specific TpCR as disclosed herein. Alternatively, thecells being endowed with antigen-specificity are the entire T-cellpopulation and the nucleic acid construct including the sequenceencoding the TpCR further includes a Foxp3 transgene that is present asan independently transcribed cistron. In another such alternative, aFoxp3 transgene is separately transfected into the T-cell population, toturn the T-cells into Treg cells. Examples provided below includestudies using murine colonoscopy, in vivo imaging andimmunofluorescence, and provide the basis for a novel cell-basedtherapeutic modality for IBD, and, by extension, for other pathologicand undesired immune responses mediated by antigen specific T effectorcells.

Various embodiments of the invention are described more specificallybelow.

-   -   The present invention is directed to a redirected regulatory T        lymphocyte (Treg cell) endowed with specificity toward a        selected target antigen or ligand, which cell comprises a        chimeric nucleic acid that encodes a chimeric receptor (CR)        polypeptide that comprises, expressed in a single, continuous        chain, an extracellular recognition region, a transmembrane        region and an intracellular signaling region, and is expressed        in the Treg cell so as to display the extracellular region on        the cell surface, wherein    -   (a) the extracellular recognition region of the chimeric        receptor is specific for the selected target antigen or ligand,        and does not comprise an MHC protein extracellular domain; and    -   (b) the intracellular region comprises a combination of T-cell        signaling polypeptide moieties which combination of moieties,        upon binding of the extracellular recognition region to the        selected target antigen or ligand, triggers activation of the        Treg cells to cause suppression of T-cell mediated immunity.

In preferred embodiments of the present invention, the extracellularrecognition region is an antibody-derived scFv domain that is specificfor a selected antigen. In another preferred embodiment, theextracellular recognition region is a member of a ligand-receptor pair,which is specific for the other member of that pair.

Preferably, the extracellular recognition region is linked to thetransmembrane region through a flexible spacer, which, more preferably,is a hinge from a molecule of the immunoglobulin family.

The intracellular signaling region preferably includes a signalingmoiety from a chain of an antigen-specific T-cell receptor, which morepreferably is one having a polypeptide region comprising animmunoreceptor tyrosine-based activation motif (ITAM). Non-limitingexamples of antigen-specific T-cell receptors are chains of the TCR/CD3complex, a TCR α, β, γ or δ chain, and the γ chain of an Ig Fc receptor(FcRγ). The chain of an antigen-specific T-cell receptor is preferablythe CD3/ζ chain or an FcRγ subunit.

The intracellular signaling region further preferably includes asignaling moiety of a costimulatory-receptor protein of a T-cell. Thecostimulatory-receptor protein is preferably selected from CD28, OX40,CD40L (gp39), 4-1BB and PD-1 (or preferably the human form or homolog ofthese costimulatory molecules). Most preferred among these is CD28 or4-1BB. In another preferred embodiment, the intracellular signalingregion includes more than one of the costimulatory-receptor proteinsignaling moieties. For example, the combination of T-cell signalingpolypeptide moieties in the intracellular signaling region may includeboth CD28 and 4-1BB. In a particularly preferred embodiment, theextracellular hinge and transmembrane regions of CD28 are used as theextracellular hinge and transmembrane regions of the chimeric receptor.

The intracellular signaling region may also include a signaling moietyfrom a cytokine receptor of a T-cell, such as the IL-2 receptor or theTGF-β receptor. The latter may help to induce the T-cell containing thechimeric receptor to adopt the characteristics of a Treg cell.

The intracellular region may also include a signal-transducing enzymethat (a) is an enzyme in the signal transduction pathway of anantigen-specific T-cell receptor or (b) is an enzyme with correspondingspecificity and activity as the enzyme of (a), derived from a non-T-celllymphocyte. Such enzyme is preferably a kinase, such as the Syk kinase.

The chimeric nucleic acid encoding the CR may also include a nucleotidesequence that encodes Foxp3 arranged such that Foxp3 is expressed by theTreg cell independently of the chimeric receptor. In other words, thechimeric nucleic acid may be bicistronic such that the Foxp3 transgeneis present as an independently transcribed cistron.

The target antigen or ligand is preferably one that is present orexpressed at a site or target tissue of an immune or inflammatoryresponse mediated by effector T-cells. The autoimmune or inflammatoryresponse may comprise an autoimmune response or disease, an allograft orxenograft response or rejection, or graft vs. host (GVH) disease. In analternative preferred embodiment, the target antigen or ligand may be anautoantigen or an antigen that is cross-reactive with an autoantigen,i.e., is also bound by an antibody that is specific to the autoantigen.The autoantigen may be a pathogenic antigen in the pathophysiology ofthe autoimmune disease.

The antigen is not necessarily an autoantigen, but can be, for example,an antigen that is part of the bacterial flora, such as LPS derived fromthe bacteria native to the colon.

The autoimmune disease or graft response and the antigen/ligand orantigens/ligands against which the Treg is specific is preferablyselected from the following group:

-   (a) inflammatory bowel disease (IBD), wherein the antigen or ligand    is one that is expressed in diseased colon or ileum;-   (b) rheumatoid arthritis, wherein the antigen or ligand is an    epitope of collagen or an antigen present in joints;-   (c) Type I diabetes mellitus or autoimmune insulitis, wherein the    antigen or ligand is a pancreatic β cell antigen;-   (d) multiple sclerosis, wherein the antigen or ligand is, for    example, a myelin basic protein (MBP) antigen or MOG-1 or MOG2-2, or    a neuronal antigen.-   (e) autoimmune thyroiditis, wherein the antigen or ligand is a    thyroid antigen;-   (f) autoimmune gastritis, wherein the antigen or ligand is a gastric    antigen;-   (g) autoimmune uveitis or uveoretinitis, wherein the antigen or    ligand is S-antigen or another uveal or retinal antigen-   (h) autoimmune orchitis, wherein the antigen or ligand is a    testicular antigen;-   (i)) autoimmune oophoritis, wherein the antigen or ligand is an    ovarian antigen;-   (j) psoriasis, wherein the antigen or ligand is a keratinocyte    antigen or another antigen present in dermis or epidermis;-   (k) vitiligo, where the antigen or ligand is a melanocyte antigen    such as melanin or tyrosinase;-   (l) autoimmune prostatitis, wherein the antigen or ligand is a    prostate antigen;-   (m) any undesired immune response, wherein the antigen or ligand is    an activation antigen or other antigen expressed on T effector cells    present at the site of the undesired response;-   (n) tissue rejection, wherein the antigen or ligand is the MHC    specific to the transplanted tissue; and-   (o) an inflammatory condition, wherein the antigen or ligand is one    that is expressed on nonlymphoid cells of the hemopoietic lineage    that participate in inflammation.

Most preferred is a Treg cell that is able to act and suppress IBD orulcerative colitis, and may be specific for an antigen associated withIBD such as carcinoembryonic antigen (CEA) or an antigen of intestinalbacterial flora such as bacterial lipopolysaccharide (LPS) or acomponent thereof, preferably a Lipid A component.

The Treg may be specific for an activation antigen expressed on Teffector cells such as CD69 or CD107a. The Treg may be specific for anantigen expressed on a dendritic cell, macrophage/monocyte, granulocyteor eosinophil present at the inflammation site.

In a preferred embodiment, the above Treg cell is specific for anantigen that is introduced exogenously to a subject to the site ortarget tissue of the immune or inflammatory response, either before,concomitantly with, or after administration of the Treg cell.

The above Treg cell preferably is one that expresses CD4 or CD8, alongwith CD25 on its surface and expresses the Foxp3 transcription factorintracellularly. The Foxp3 transcription factor may be expressed in thecell endogenously (i.e., from the cells' own Foxp3 gene); thisexpression is enhanced by exposure of cells to TGF-β or another cytokinethat induces Foxp3 expression and induces a Treg phenotype in T-cells.In another embodiment of the above Treg cell, Foxp3 is expressed from anucleic acid that has been introduced into the cell exogenously (i.e.,transduced) as a recombinant nucleic acid expression construct encodingFoxp3 and regulating its expression. The above Treg may be obtained froma mammalian subject prior to introduction of the chimeric nucleic acidand prior to stimulation that induces Foxp3 expression or prior totransducing the exogenous Foxp3-encoding construct. The chimeric nucleicacid encoding the chimeric receptor and the nucleic acid constructencoding Foxp3 may be co-transduced into the cell. In one embodiment,co-transduction is achieved using a bicistronic vector that includes, ina single vector, a sequence of (i) the chimeric nucleic acid encodingthe chimeric receptor and (ii) the nucleic acid construct encodingFoxp3, under the control of a common (or separate) promoter andregulatory sequences.

The above Treg cells may be enriched or purified from a mixed populationof lymphocytes or T-cells on the basis of the Treg cells' expression ofCD4 (or CD8) and CD25 and/or Foxp3. The cell may be subjected to thefollowing treatment:

-   (a) exposure ex vivo of:    -   (i) peripheral blood mononuclear cells,    -   (ii) peripheral blood lymphocytes,    -   (iii) T-cells enriched or purified from (i) or (ii), or    -   (iv) a subset of T-cells enriched or purified from (iii); to an        amount of TGF-β or other Treg-inducing cytokine or agent that is        effective to convert T-cells to a Treg phenotype and to induce        expression of Foxp3; and-   (b) optionally, culturing and expanding the exposed cells of step    (a).    Preferred Treg cells comprise the above cell that has been    transduced with an expression vector encoding Foxp3.

Also provided herein is an immunoregulatory pharmaceutical compositionfor suppressing a T effector cell-mediated immune/inflammatory responseor treating a T effector cell-mediated immune/inflammatory disease orcondition, comprising a redirected Treg as described above and apharmaceutically and immunologically acceptable carrier, excipient ordiluent.

This invention is also directed to a method for producing the aboveredirected Treg that expresses a chimeric receptor as described. Thismethod preferably comprises:

-   (a) obtaining from a subject and, optionally, enriching or isolating    and propagating, a population of lymphocytes or T-cells;-   (b) inducing the Treg phenotype in these lymphocytes by suitably    stimulating or activating the cells by exposure to TGF-β or another    cytokine or agent that induces Foxp3 expression and a Treg    phenotype;-   (c) before or after step (b), transducing the cells ex vivo with an    expression vector encoding the chimeric receptor to be expressed in    the Treg; and-   (d) optionally, growing or expanding in vitro the cells obtained as    above.

In another embodiment, this method comprises:

-   (a) obtaining from a subject and, optionally, enriching or isolating    and propagating, a population of lymphocytes or T-cells;-   (b) transducing the cells ex vivo with a vector encoding the    chimeric receptor;-   (c) before, after, or concomitantly with step (b), transducing the    cells ex vivo with a recombinant nucleic acid expression construct    encoding Foxp3; and-   (d) optionally, growing or expanding in vitro the cells obtained as    above.

This invention further is directed to a method of suppressing undesiredactivity of T effector cells in mediating an immune or inflammatoryresponse, comprising delivering to a population of T effector cells tobe suppressed (or to a site where such T effector cells are present) anamount/number of redirected Tregs as above, effective to suppressactivity of the T effector cells.

Also intended is a method of suppressing undesired activity of Teffector cells as indicated above, which method comprises delivering toa population of T effector cells to be suppressed (or to a site wheresuch T effector cells are present) an amount/number of redirected Tregsproduced according to the above methods that are effective forsuppressing the T effector cell activity.

This delivering is preferably in vivo. The redirected Treg cells aredelivered by injection or infusion to a subject in whom the T effectorcell activity is to be suppressed, preferably by a route selected fromintravenous, intramuscular, subcutaneous, intraperitoneal,intra-articular, intrathecal, intraluminal, intracerebroventricularly,rectal, and topical. In one embodiment, the Treg cells are deliveredregionally or locally to a site of inflammation.

The above method is intended for use in situations wherein the Teffector cells mediate an autoimmune inflammatory response or disorder,rejection of a transplant or GVH disease.

In one embodiment, the method for treating or ameliorating symptoms of adisease or condition in a subject that is mediated by undesired activityof T effector cells comprises administering to the subject in needthereof an effective amount/number of Treg cells as described above, ora pharmaceutical composition described above, wherein the targetrecognition domain of the redirected Treg cells is specific for anantigen/ligand present in the subject in the vicinity of the T effectorcells so that, upon recognizing and binding the antigen, theseredirected Treg cells are activated to secrete suppressive cytokinesthat suppress the T effector cells in an antigen-nonspecific manner. Asnoted above, the Treg cell activation occurs in a manner that is notrestricted by the MHC and does not require costimulation by a ligand forthe costimulatory signaling protein.

Also included is a method for treating or ameliorating symptoms of adisease or condition in a subject that is mediated by undesired activityof T effector cells, the method comprising: (a) producing redirectedTreg cells using the production methods described above; (b)administering to the subject in need thereof an effective amount/numberof these Treg cells, thereby treating or ameliorating symptoms of thedisease or condition.

Stated more generally, the invention is directed to a method forsuppressing a T effector cell-mediated immune/inflammatory process in asubject in need thereof, comprising administering to the subject aneffective amount/number of redirected Treg cells that express on theirsurface an antigen-specific chimeric receptor that includes portionsthat activate Treg cells upon contact with the antigen for which thereceptor is specific, the antigen being one that is present in thevicinity of the immune/inflammatory activity. The disease or conditionto be treated or ameliorated is preferably: (a) IBD; (b) rheumatoidarthritis; (c) Type I diabetes mellitus or autoimmune insulitis; (d)multiple sclerosis; (e) thyroiditis; (f) gastritis; (g) uveitis oruveoretinitis; (h) orchitis; (i)) oophoritis; (j) psoriasis; (k)prostatitis; (l) encephalomyelitis; (m) vitiligo; (n) rejection of amismatched cell, tissue or organ graft; or (o) GVH disease.

The present method is used to inhibit the rejection of transplantedcells, tissue, or an organ (alto- or xeno-) that is, for example,mismatched for a major and/or one or more minor histocompatibilityantigens. In the case of GVH disease, the recipient generally hasreceived a transplant of allogeneic, semi-allogeneic ornon-MHC-mismatched bone marrow cells or enriched or isolatedhematopoietic stem cells that are responsible for mediating pathogeniceffects.

The present invention is further directed to the novel chimeric DNA thatcan be used to produce the redirected T-cells described above, as wellas to the chimeric receptor protein encoded thereby. Such chimeric DNAcomprises:

a first DNA segment encoding an extracellular recognition regionspecific for a selected target antigen or ligand, which does notcomprise an MHC protein extracellular domain, the selected targetantigen or ligand being one that is present or expressed at a site ortarget tissue of a pathogenic or undesired immune response mediated byeffector T-cells;

a second DNA segment encoding a transmembrane region; and

a third DNA segment encoding an intracellular signaling regioncomprising a combination of T-cell signaling polypeptide moieties, whichcombination of moieties, upon transfection of the chimeric DNA into aregulatory T lymphocyte (Treg cell) and binding of the extracellularrecognition region to the selected target antigen or ligand thereof,triggers activation of the Treg cells to cause suppression of T-cellmediated immunity, which chimeric DNA, upon transfection into a Tregcell, expresses the extracellular recognition region, the transmembraneregion and the intracellular signaling region in one single, continuouschain on the surface of the transfected cell such that the transfectedTreg is triggered to activate and cause suppression of T-cell mediatedimmunity when the expressed extracellular recognition region binds toits selected target antigen or ligand.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic diagram of TNP-specific TPCR structure. (A) Schematicpresentation of the TNP-specific chimeric receptors. The TNP-specific CRencompasses a scFv derived from the anti-TNP mAb, Sp6. In the tripartiteconfiguration, the scFv is joined in tandem to a short portion of CD28(lacking the ligand-binding site) of the extracellular and including thetransmembrane, and cytoplasmic domains fused to the FcRγ ITAM domain.(B) Chimeric receptor transgene constructs. Constructs used to generatethe transgenic mice were placed under the control of the human CD2promoter/enhancer that directs expression only in T and NK cells. CYTindicates cytoplasmic domain; H, hinge domain; L, immunoglobulin leader;LCR, locus control region; P, promoter; pL, plasmid sequence; TM,transmembrane domain; VH and VL, immunoglobulin heavy and light-chainvariable domains, respectively; ACD28, truncated CD28 containing part ofthe extracellular and the transmembrane domain, and lacking thecytoplasmic signaling moiety.

FIG. 2: Flow cytometry results of Foxp3 staining of TNP-specific Tregs.Splenocytes isolated from WT and TNP-Tg mice were stained forintracellular Foxp3 and for TNP-specific chimeric receptor usingantiidiotypic antibody to the Sp6 scFv. Representative flow cytometryanalyses are shown for an individual mouse out of five tested mice.Percentages indicate double-stained cells.

FIG. 3: Graph showing the ratio of CD4+CD25+ cells to CD4+ cells insplenocytes of wildtype and Tg mice. The groups are: wildtype mice, miceTg for a chimeric receptor specific for an “irrelevant” antigen, ErbB2(also referred to as ErbB2-Tg mice), TNP-Tg mice that have beentransfected with a vector lacking the transgenic costimulatory CD28domain (also referred to as TNPΔCD28-Tg mice), and TNP-Tg. TNP-Tg Tregsfully express the TNP-specific TpCR

FIG. 4: Graph (left) showing Foxp3+/CD4 cell ratio in wildtype,ErbB2-Tg, TNPΔCD28-Tg, and TNP-Tg mice. Flow cytograms (right) showingsplenic Foxp3 expression. Results compare wildtype, ErbB2-Tg, TNP-Tg andTNPΔCD28-Tg mice.

FIG. 5: Flow cytograms showing Foxp3 staining in sorted wildtype,ErbB2-Tg, TNP-Tg and TNP-ΔCD28-Tg CD4+CD25+ effector T-cells.

FIG. 6: Graph (left) showing ratio of Foxp3+ to CD25+/Foxp3+ cells. Flowcytogram (right) showing co-staining of Foxp3 and CD25. Results comparewildtype, ErbB2-Tg, TNP-Tg and TNP-ΔCD28-Tg splenocytes.

FIG. 7: Graph showing percentage of Foxp3+ splenocytes in the totalCD3+T-cell population following induction of TNBS colitis. Spleniclymphocytes were isolated from WT and TNP-Tg mice prior to or 48 hoursfollowing induction of TNP colitis, and double-stained with anti-Foxp3and anti-CD3 antibodies.

FIG. 8: Graph showing percentage of Foxp3+ lymphocytes extracted fromcolonic lamina propria following induction of TNBS colitis. Lymphocyteswere isolated from WT and TNP-Tg mice prior to and 48 hours followinginduction of TNP colitis, and double-stained as in FIG. 7. Thepercentage of Foxp3+ lymphocytes in the CD3+ population is presented asthe average Foxp3/CD3 ratio ±s.d. of each five-mouse group. Data shownare averages of two independent experiments performed. Differences inratios between naïve and colitis-induced TNP-Tg mice were significant(P<0.05).

FIG. 9: Series of 6 graphs showing stimulation of proliferation ofredirected Tregs (left) and T effector cells (right) by anantigen-nonspecific stimulus (anti CD3 and anti CD28 mAbs) andantigen-specific (TNP) stimulus.

FIG. 10: Graph showing polyclonal activation with Concanavalin A (Con A)of co-cultures of Tregs and T effectors cells (and control cultures ofindividual cell populations)

FIG. 11: Graphs showing Specific activation of TNP-Tg Tregs and theirsuppression of effector T-cells requires TNP and CD28-co-stimulation. Inthe left panel, specific (TNP) activation of Tregs is shown. WT orTNP-Tg Tregs (5×10⁴) were cocultured with WT or TNP-Tg Teff (5×10⁴) inthe presence of irradiated, T-cell depleted, TNPylated splenic APC(1.5×10⁵). Teff proliferation was measured after 48 hours by³H-Thymidine incorporation. Right panel: TNP-loaded APCs as stimulus.

FIG. 12: Graph showing dose-response of TNP-specific stimulation of Tregcell+T effector cell cocultures. APCs were TNP-modified stimulator P815mastocytoma cells, which do not express B7 (P815-TNP) or TNP-modifiedP815 cells into which the B7 gene was stably transfected (B7-TNP).

FIG. 13: Graph showing Specific activation of TNP-Tg Tregs and theirsuppression of effector T-cells requires TNP and CD28-co-stimulation. Toestablish whether costimulatory signaling is required for TNP-Tg Tregactivation, coculture experiments were repeated using as APC irradiatedP815 mastocytoma cells (1.5×10⁵) that were either stably transfected (ornot) with B7 cDNA. Teff cell proliferation was measured after 48 hoursby ³H-Thymidine incorporation. Each group was cultured in triplicate andthe experiment was repeated three times. The data shown represent mean(+s.d.) of triplicate cultures of a representative experiment.Differences in stimulation index between Teff+WT Tregs and Teff+TNP-TgTregs were significant (P<0.01).

FIG. 14: Photograph of colon of wildtype mice (left) and TNP-Tg mice(right) four days after induction of high-dose TNBS colitis byintrarectal instillation of TNBS at day 0.

FIG. 15: Mortality curve of wild-type (WT), TNP-Tg, ErbB2-Tg andTNP-ΔCD28-Tg mice following induction of TNBS colitis by intrarectalinstillation of TNBS at day 0.

FIG. 16: Photomicrograph of stained tissue (H&E, 40×) of the colons fromFIG. 14.

FIG. 17: Mortality curve of WT, TNP-Tg, ErbB2-Tg and TNP-ΔCD28-Tg micefollowing induction of colitis with oxazolone (OXA; a hapten/antigenthat is distinct from TNP). These results serve as a control for Tregspecificity in the experiment the results of which are shown in FIGS.14-16. Colitis was induced using the unrelated hapten, oxazolone, whichwas intrarectally instilled in similar strains of mice (n=10).

FIG. 18: Flow cytograms of Foxp3 staining of wildtype, ErbB2-Tg,TNP-ΔCD28-Tg and TNP-Tg T effector cells after a week of culture in thepresence of the following “stimuli” (across top): anti-CD3, TGF-β,anti-CD3+TGF-β, TNP, or TNP+TGF-β.

FIG. 19: Graphs showing mortality or survival rate of wildtype micesubjected to induction of moderate (left panel) and severe (right panel)TNBS colitis following adoptive transfer of the following Tregpopulations: WT, ErbB2-Tg, TNP-Tg and TNP-ΔCD28-Tg.

FIG. 20: Graph showing Wallach Colitis Severity Score of wildtype micesubjected to induction of TNBS colitis after adoptive transfer of cellsfrom WT, ErbB2-Tg or TNP-Tg donors. TNBS colitis was induced in WT mice(n=8) on day 0. After 16 hours, Tregs (1×10⁵) from TNP-Tg, ErbB2-Tg orWT mice were adoptively transferred to the recipient mice. Eachexperiment was repeated three times. The data shown represent theaverage of a representative experiment.

FIG. 21: Photograph of excised colons of wildtype mice in which TNBScolitis was induced, following adoptive transfer of wildtype, ErbB2-Tgand TNP-Tg mice in the experiment described in FIG. 20.

FIG. 22: Photomicrograph of stained colon tissue sections (H&E, 40×)from wildtype mice with TNBS colitis after adoptive transfer of Tregsfrom the following donors: (A) wildtype (B) ErbB2-Tg and (C) TNP-Tg.Panel D shows normal control colon.

FIG. 23: Localization of Tregs in the colon. Flow cytograms offluorescent staining Tregs labeled with the intracellular dyecarboxyfluorescein diacetate succinimidyl ester (CFSE) in the coloniclamina propria of naïve mice or mice with TNBS-colitis. Labeled Tregswere intraperitoneally injected 24 hours following induction of TNBScolitis. Lymphocytes from lamina propria were obtained 16 hrs afteradoptive transfer of 10⁶ wildtype or TNP-Tg Tregs to indicatedrecipients. CFSE-labeled Tregs were 9-fold more abundant in diseasedcolons. Data shown represent the percentages of CFSE-positive cells inthe corresponding gates of one representative mouse of each four-mousegroup. Each experiment was repeated twice.

FIG. 24: Localization of Tregs in the colon. In-vivo imaging of WT micereceiving DiR-labeled wildtype and TNP-Tg Tregs (1×10⁶) 16 hoursfollowing induction of TNP colitis (n=3). Mice were subjected to a wholebody imaging (IVIS® 100 Series Imaging System) at 12 hour intervals. Asingle representative mouse out of three in each group is shown at alltime points. Two independent experiments were performed, with similarresults. (

FIG. 25: Localization of Tregs in the colon. In-situ fluorescentmicroendoscopic (Cell Vizio) evaluation of CFSE-labeled Tregsaccumulating at the colonic pre-luminal mucosal layer. The experimentaldesign is identical to the one described in FIG. 23. The figure showsrepresentative frames taken 48 hours following adoptive transfer. Eachgroup consisted of four mice, and each experiment was repeated twice.

FIG. 26: Intrarectal administration of TNBS results in TNP-TgTreg-mediated protective effect from oxazolone colitis. (a) Mortalityrates of wildtype and TNP-Tg mice administered oxazolone±low doses ofTNBS, 1 week following presensitization with oxazolone only. (b) Murinecolonoscopy images of representative WT and TNP-Tg mice. (c) Macroscopicappearance of representative colons from various mouse groups. (d)Microscopic appearance of colons shown in c. (e) Adoptive transfer ofTregs (Tr) to oxazolone (O) pre-sensitized mice induced one week laterwith oxazolone (O) colitis in the presence of low dose of TNBS (T). WTor TNP-Tg Tregs were administered to mice (n=8) 16 hours after theinduction of colitis.

FIG. 27 shows 8 schematic drawings of T cells which are transduced witha retroviral vector that carried one of 8 CR constructs that include thedetectable fluorescent protein, eGFP. Those depicted in the lower halfof the Figure are bicistronic constructs that encode a fusion of GFP andthe transcription factor Foxp3. Light and fluorescence microscopy wereused to follow expression of the GFP in the cytoplasm of nucleus of thetransduced cells. The constructs are labeled as follows (where “TPCR”refers to “tripartite chimeric receptor” even though, some of these CR'swere “more” than tripartite).

-   -   a. TNP-TPCR: extracellular recognition region comprised an scFv        of a TNP-specific mAb.    -   b. MD2-TPCR: extracellular recognition region comprised an LPS        binding fragment or motif of the human MD2 protein, an LPS        co-receptor (that interacts with TLR4 receptors). The fragment        of MD2 corresponds to residues 120-132 of SEQ ID NO:5. The        sequences of such chimeric nucleic acids used here are SEQ ID        NO:8 and 9.    -   c. CD14-TPCR: extracellular recognition region comprised an LPS        binding fragment of the human CD14 protein, a known LPS        receptor. The fragment of CD14 corresponds to residues 100-119        of SEQ ID NO:4. The sequences of such chimeric nucleic acids        used here are SEQ ID NO:6 and 7.    -   d. MD2-CD14-TPCR: the extracellular recognition region comprised        both the MD2 fragment and the CD14 fragments described above.        The sequences of such chimeric nucleic acids used here are SEQ        ID NO:10 and 11.        Each of the constructs encoded as stimulatory and costimulatory        moieties, tandemly linked sequences encoding CD28 and FcRγ.        Results are shown as side-by-side light and fluorescence        micrographs.

FIG. 28. An annotated nucleotide sequence (SEQ ID NO:1) and amino acidsequence (SEQ ID NO:2) of a TNP-specific tripartite CR as used herein.The annotations include the origin of the regions (scFv, here the “Sp6”mAb), the “CD28” region, and the FcRγ regions (indicated as “GAMMA”), aswell as restriction sites, leader sequence, etc. The mature proteinbegins at amino acid residue 23.

FIG. 29. An annotated nucleotide sequence (SEQ ID NO:3) of a pBulletplasmid that includes a CR-encoding construct that comprises anucleotide sequence encoding the scFv of mAb HB 9081 (i.e., produced bya hybridoma given ATCC Accession No. HB9081) fused to C28/FcRγ. This mAband, hence, the scFv, is specific for LPS. Annotations show variousrestriction enzyme recognition sites, the leader sequence, and plasmidsequences.

FIG. 30A-30B. FIG. 30A is an annotated amino acids sequence of HumanCD14 (SEQ ID NO:4). See GenBank Accession No. P08571. A signal sequenceand an LPS-binding motif (residues 100-119) are noted. This proteinserves as an LPS receptor on cells. FIG. 30B is an annotated amino acidssequence of Human MD-2 protein (SEQ ID NO:5). See GenBank Accession No.NP_(—)056179. A signal sequence and an LPS-binding motif (residues120-132) are noted. This LPS-binding protein interacts with TLR-4 as aco-receptor.

FIG. 31A-31B. FIG. 31A is an annotated nucleotide sequence (SEQ ID NO:6)showing the nucleotide sequence of a Chimeric Receptor comprising CD14motif-CD28-FcRγ. FIG. 31B is an annotated nucleotide sequence (SEQ IDNO:7) showing the nucleotide sequence of a chimeric, bicistronicreceptor: CD14 motif-CD28-FcRγ-IRES-GFP-Foxp3. Also shown is the aminoacid sequence (single letter code) of the CD14 motif (residues 110-119of SEQ ID NO:4). Annotations show various restriction sites, beginningsand ends of protein regions, IRES region, etc.

FIG. 32A-32B. FIG. 32A is an annotated nucleotide sequence (SEQ ID NO:8)showing the nucleotide sequence of a Chimeric Receptor comprising MD2motif-CD28-FcRγ. FIG. 32B is an annotated nucleotide sequence (SEQ IDNO:9) showing the nucleotide sequence of a chimeric, bicistronicreceptor: MD2 motif-CD28-FcRγ-IRES-GFP-Foxp3. Also shown is the aminoacid sequence of the MD2 motif (residues 120-132 of SEQ ID NO:4.Annotations show various restriction sites, beginnings and ends ofprotein regions, IRES region, etc.

FIGS. 33A and 33B. FIG. 33A is an annotated nucleotide sequence (SEQ IDNO:10) showing the nucleotide sequence of a Chimeric Receptor comprisingMD2 motif-CD14 motif-CD28-FcRγ. FIG. 33B is an annotated nucleotidesequence (SEQ ID NO:11) showing the nucleotide sequence of a chimeric,bicistronic receptor: MD2 motif-CD14 motif-CD28-FcRγ-IRES-GFP-Foxp3.Also shown is the amino acid sequence of the MD2 motif (residues 120-132of SEQ ID NO:4) and the amino acid sequence of the CD14 motif (residues100-119 of SEQ ID NO:3). Nucleotides 106-148 of SEQ ID NO:11 (doubleunderlined) encode a flexible linker (14 amino acids, SEQ ID NO:12, alsodouble underlined). Annotations show various restriction sites,beginnings and ends of protein regions, IRES region, etc.

FIG. 34 is an annotated nucleotide sequence (SEQ ID NO:13) showing thenucleotide sequence of a Chimeric Receptor comprising MD2-CD28-FcRγ (SEQID NO:13). Also shown is the amino acid sequence of the full length MD2protein (SEQ ID NO:4). The LPS-binding region of this amino acidsequence is underscored. Annotations show various restriction sites,beginnings and ends of protein regions, etc.

FIG. 35 is an annotated nucleotide sequence (SEQ ID NO:14) showing thenucleotide sequence of a chimeric, bicistronic receptor:MD2-CD28-FcRγ-IRES-GFP-Foxp3. Also shown is the amino acid sequence ofthe full length MD2 protein (SEQ ID NO:4). The LPS-binding region ofthis amino acid sequence is underscored. Annotations show variousrestriction sites, beginnings and ends of protein coding regions, IRES,vector sequence, etc.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

“Regulatory T lymphocyte” or “Treg cell” or “Treg,” as used in thepresent specification and claims are synonymous and are intended to haveits standard definition as used in the art. Treg cells are a specializedsubpopulation of T cells that act in a “regulatory” way to suppressactivation of the immune system and thereby maintain immune systemhomeostasis and tolerance to self-antigens. Tregs have sometimes beenreferred to suppressor T-cells. Treg cells are characterized byexpression of the forkhead family transcription factor Foxp3 (forkheadbox p3). They may also express CD4 or CD8 surface proteins. They usuallyalso express CD25. As used in the present specification and claims, andunless otherwise specified, Tregs include natural Tregs and induced oradaptive Tregs and Tregs that have been created using recombinant DNAtechnology. Naturally-occurring Treg cells (CD4+CD25+Foxp3+) arise likeall other T cells in the thymus. In contrast, adaptive Treg cells (alsoknown as Trl cells or Th3 cells) may originate during a normal immuneresponse. Antigen-specific activation of human effector T-cells leads toinducible expression of Foxp3 in a subgroup of the activated effectorcells, and this subgroup can develop a regulatory (Treg) phenotype. Oneway to induce Tregs is by prolonged exposure of T effector cells toTGF-β. T-cells may also be converted to Treg cells by transfection ortransduction of the Foxp3 gene into a mixed population of T-cells. AT-cell that is caused to express Foxp3 adopts the Treg phenotype andsuch recombinant Tregs are also defined herein as “Tregs”.

“Redirected Treg” is intended to be a comprehensive term for Tregscarrying a chimeric receptor (CR) as described and claimed herein whichconfers on the cells the ability to bind to and be activated by a targetantigen or ligand that is different from that to which a Treg populationmay have been previously specific (as controlled by its endogenousantigen-specific TCR). Redirected Tregs are “MHC-independent” and“non-MHC restricted” in the process of their activation and in theiractions as they do not require association of a peptide derived fromtheir target antigen or ligand with MHC in order to recognize it.However, for special purposes, it may be possible to design a redirectedTreg that recognizes a specific epitope of an MHC molecule per se, e.g.,functioning as a transplantation antigen. In such a case theseredirected Tregs are still non-MHC restricted.

The term “selected target antigen or ligand” means a molecule to whichthe extracellular recognition region of the redirected Treg is intendedto bind so as to activate that Treg. If the selected target is anantigen, then an antibody can be raised against it and the bindingregions of such an antibody used to construct the extracellularrecognition region of the redirected Treg. If the target molecule is amember of a receptor/ligand pair (defined below), then the other memberof that pair can be used as part of the extracellular recognition regionof the redirected Treg. Generally, in designing a redirected Treg foruse according to this invention, the intended target tissue or sitewhere the Treg is to be employed is first identified, and then, anantigen or ligand that is present on or near this intended target tissueor site is identified. An antibody or ligand/receptor that binds theretois then identified, or, if necessary, created or constructed for use onthe redirected Treg.

The term “ligand” as used herein, and particularly as part of the term“target antigen or ligand” or the term “receptor/ligand pair” refers toa molecule that is able to bind to and form a complex with anotherbiomolecule to serve a biological purpose. Often the binding partner ofa ligand is called a receptor so that the two binding partners aretermed a “receptor/ligand pair.” For the purpose of the presentspecification and claims, the term “receptor,” when used in the sense ofa “receptor/ligand pair,” has a broader meaning than, for example, atypical definition of a “receptor” as a protein in or on a cell thatbinds to a specific ligand. It is rather intended to mean any bindingpartner for a ligand. Either member of a binding pair can be consideredthe “ligand” while the other member is considered the “receptor.” Thus,a classical receptor may qualify as a “ligand” when used herein in theterm “target antigen or ligand” as it is one member of a receptor/ligandbinding pair. For example, IL-2 can be a ligand because it binds to andforms a complex with another biomolecule, i.e., an IL-2 receptor(IL-2R), to serve a biological purpose. However, IL-2R is also a“ligand” because it is a molecule that binds to and forms a complex withanother biomolecule, i.e., IL-2, to serve a biological purpose. Thus,under the present usage, if IL-2R is considered a ligand in theIL2R/IL-2 binding pair, IL-2 may be considered the receptor, and viceversa.

A “chimeric receptor,” as used in the present specification and claims,is a recombinant polypeptide that includes an extracellular recognitionregion that is derived from one molecule and at least one intracellularsignaling moiety that is derived from a different molecule. In thatsense it is chimeric.

A “chimeric nucleic acid” is a recombinant polynucleotide that includesa sequence that encodes a chimeric receptor.

The terms “recombinant” or “recombinantly” when applied to apolynucleotide, polypeptide or cell means that the molecule or cell ismade using genetic engineering techniques and would not exist but forthe hand of man.

The term “T-cell signaling polypeptide moiety” means that portion of amolecule endogenous to a T-cell that mediates signaling. It may be aportion of a T-cell receptor molecule that mediates signaling, or adownstream signal-transducing enzyme or a portion thereof that mediatessignaling, i.e., that has enzymatic activity.

The term “antibody-derived scFv domain” means a single-chain antibody inwhich the V_(L) of a specific antibody is linked to the V_(H) thereof bya flexible linker or spacer.

The term “an MHC protein extracellular domain” refers to the disclosureof Meal D J et al. (Proc Nall Acad Sci USA 2005; 102:11817-22),discussed below, and Jodi M D et al., Nat. Biotechnol., 2002,20:1215-1220. These publications describe Treg cells redirected againstT-cells in a murine system. They used the class II MHC Is α and Is βchains as extracellular regions of two separate chimeric receptors foruse in a redirected Treg. The term MHC protein extracellular domain isdefined so as to encompass what was used in the Meal et al. and Jodi etal. publications and any analogs or fractions thereof that would havebeen obvious to a person of ordinary skill in the art to substitute forsuch domains for the purpose disclosed by Meal et al. and by Jodi etal., i.e., to cause binding of the redirected Treg to a T lymphocytespecifically directed to a particular autoantigen.

The term “flexible spacer” means any flexible peptide moiety that willfacilitate the functionality of the extracellular recognition region.When this region is not rigidly attached to the transmembrane region,but is allowed some degree of flexibility with respect to the cellmembrane, the ability of the recognition region to recognize and bind toits target antigen or ligand is facilitated. Small neutral amino acids,such as glycine and serine, confer such flexibility. Examples areGly₄Ser and Gly₄Ser₃.

The “immunoglobulin superfamily” (Igs) means the large group of cellsurface and soluble proteins that are involved in the recognition,binding, or adhesion processes of cells. Molecules are categorized asmembers of this superfamily based on shared structural features withimmunoglobulins (also known as antibodies); they all possess a domainknown as an immunoglobulin domain or fold. Members of the Igs includevarious cell surface antigen receptors, co-receptors and co-stimulatorymolecules of the immune system, molecules involved in antigenpresentation to lymphocytes, cell adhesion molecules and certaincytokine receptors. They are commonly, though not exclusively,associated with roles in the immune system.

The term “hinge” when referring to a region of a molecule of the Igsmeans the region between the C_(H)1 and C_(H)2 domains consisting of asmall number of amino acids. The hinge is flexible and allows thebinding region to move freely relative to the rest of the molecule. Atthe hinge region are the disulfide bridges which link the two dimers,creating the tetramer structural unit. Examples of such immunoglobulinhinge sequences may be found in U.S. Pat. No. 6,165,476, which isincorporated herein by reference.

The term “antigen-specific receptor of a T-cell” refers to a receptorthat is found on a T-cell that is antigen-specific, i.e., naturally hasan extracellular region that binds specifically to a particular antigenin preference to another. Examples of such antigen-specific receptors ofa T-cell are the TCR α, β, γ or δ chains, the TCRαβ dimer and TCR dimer.

The term “TCR/CD3 complex” is sometimes called the “TCR complex.” CD3 isa protein complex composed of four chains in mammals (CD3γ, CD3δ and twoCD38 chains), that associate with molecules known as the T cell receptor(TCR; see above) and with the ζ-chain and η-chain (as homo- orheterodimers) to generate an activation signal in T lymphocytes. Theintracellular tails of these CD3 molecules contain a single conservedmotif known as an “immunoreceptor tyrosine-based activation motif” orITAM for short, which is essential for the signaling capacity of theTCR. The CD3-γ, -δ, and -ε chains and the ζ- and η-chains, also known asCD3-ζ and CD3-η chains, together with the TCR, form what is known as theT cell receptor complex.

The term “T-cell costimulatory-receptor protein” means a receptor of theT-cell that provides a costimulatory signal. During the activation of Tcells, costimulation is often crucial to the generation of an effectiveimmune response. T cells require two signals to become fully activated.A first, antigen-specific, signal is provided through the T cellreceptor/CD3 complex. A second signal, the costimulatory signal, isantigen-nonspecific and is provided by costimulatory molecules expressedon the T cell membrane. Examples of T-cell costimulatory-receptorproteins are CD28, OX40, CD40L, 4-1BB and PD-1.

The present invention is based on the conception that regulatory T-cells(Treg) that have been modified to possess antibody-type antigenspecificity, can be harnessed to suppress T effector cells function invivo. The action of these Treg cells is mediated in anantigen-nonspecific manner, primarily by release of suppressivecytokines in the vicinity where the Tregs are activated or stimulated byan antigen recognized by their TpCR. Once activated, Tregs can suppressbystander T cell responses. Thus, transfer of these cells that have beenengineered to express the CRs (as described herein) to a subject in whomit is desired to suppress a T effector cell response and its attendantor consequent inflammation, and their delivery to, and activation at,the site of such inflammatory activity, results in therapeutic effects.

Thus, preferred target diseases or conditions for this invention areautoimmune diseases, more preferably, organ-specific, T cell-mediatedautoimmune diseases. Other examples of undesired immune responsivenessto be targeted herein are graft rejection of solid tissue and organgrafts as well as grafts of suspended cells (e.g. bone marrow (BM)transplants or hemopoietic stem cell (HSV) transplant). Another targetdisease is graft-vs-host disease (GVH) that is a common consequence of amismatched BM or HSV transplant. An additional condition targeted bythis invention is transplant rejection (e.g., of a mismatched kidney)where the recipient's immune effector cells reject the graft.

Because the Foxp3 transcription factor (a member of the forkhead family)appears to be essential for Treg development and function, and is adistinctive marker for these cells (along with CD4 and CD25), thepresent invention provides methods for producing Tregs, as well asproviding the Tregs produced by those methods, that are based oninduction of Foxp3 in T-cells in a process of driving cells along thepathway to Treg status.

In another embodiment, DNA encoding Foxp3 is transduced or transfectedinto T-cells using any suitable expression vector as a delivery vehiclein a process to drive these cells to become Tregs. Further support forthis conception is found in reports that prevention of Foxp3 expressionin vivo results in animals with a propensity for development ofautoimmune and lymphoproliferative disorders (Sakaguchi S, et al., JImmunol 1995; 155:1151-64; Hori S et al., Science 2003; 299:1057-61;Khattri R, et al., supra; Fontenot J D et al., Nat Immunol. 2003;4:330-6.).

The starting population can be total PBL, T-cells that have beenenriched or isolated from PBL, or CD4+T-cells that have been enriched orisolated from such T-cells (either expressing CD25 or not). These cellscan be redirected by transducing the TpCR prior to, concomitantly with,or after transducing DNA encoding Foxp3 DNA, preferably in the form of aFoxp3 expression vector. Walker M R. et al., 2005, Proc Natl Acad SciUSA. 102:4103-8, have shown that antigen-specific human CD4+CD25+ Tregcells can be generated de novo from CD4+CD25− cells. The advantage ofthe present invention over the approach described by Walker et al. isthat the antigen-specificity and Treg activation requirements areindependent of the MHC. This important improvement makes isolation andactivation of antigen-specific Tregs simpler and allows for therapeuticmethods (described below) in which the antigen can be convenientlyadministered together with the transferred Treg cells to a desired site,such as an inflammatory site, exemplified by the colon in IBD.

Naturally-Occurring vs. Inducible Tregs

“Classical” naturally-occurring Tregs are thymus-derived, express highlevels of Foxp3 and suppress activation of effector lymphocytes.Antigen-specific activation of human effector T-cells leads to inducibleexpression of Foxp3 in a subgroup of the activated effector cells, whichsubgroup can develop a regulatory (Treg) phenotype. These inducedregulatory T-cells can suppress (independently of cell contact) freshlyisolated effector cells (Walker M R., et al., 2005, supra; Walker M R.,et al., 2003, J Clin Invest. 112:1437-43). In mice, both in vitro and invivo induction of Tregs is achieved by prolonged exposure of effectorcells to TGF-β (Wan Y R, et al., 2005, Proc Natl Acad Sci USA.102:5126-31; Mantini M C, et al., 2004, J Immunol. 72:5149-53; Mantiniet al., 2006, supra). This small, peripherally generated population ofinducible Tregs are believed to play a central role in regulating andcontaining ongoing immune responses just as the lack of Treg inductionis associated with a propensity for autoimmunity.

Genetic Manipulation of CD4+CD25+T Regulatory Cells

According to the present invention, approaches that specificallyredirect regulatory T-cells to suppress the activity of pathologicalT-cells are beneficial in inflammatory conditions by facilitatinglocalization of Tregs to inflammatory sites and their specificactivation by inflammation-associated antigens. When specificallyactivated in inflammatory lesions, such Tregs are expected to attenuateinflammatory disease by suppressing pathogenic effector T lymphocytes inan antigen-nonspecific, MHC-unrestricted, manner.

The MHC-independent activation and action of Treg cells according to thepresent invention is an important advantage. Such action is contrastedwith the report of Meal D J et al. (Proc Natl Acad Sci USA 2005;102:11817-22) of a study of experimental allergic encephalomyelitis(EAE) which described CD4+25+ Treg cells redirected against myelin basicprotein (MBP) epitope 89-101-reactive T cells by a CR that included theMBP epitope linked to MHC class II protein. By enforcing the interactionbetween a Treg cell and the autoreactive T cells directed against MBPepitope 89-101, the Treg activity is antigen-specifically focusedagainst the autoreactive T-cells. Such a model requires some degree ofMHC-dependency as a single CR can only have domains of a single MHC andthus can only be used for patients with that HLA characteristic.

In contrast, the Treg cells of the present invention act to suppresspathogenic T effector cells in an MHC-independent manner, making themmore advantageous for treating autoimmune/inflammatory conditionsbecause they can target common target antigens shared among manyindividuals. In the model of Meal et al., supra, Tregs acted by MHC- andantigen-restricted engagement. As such these Tregs, which express theligand that is recognized by the TCR of the autoreactive T-cells, arestimulated by such interaction and suppress the effector cells. However,as a clinical approach this suffers from the disadvantage that it wouldrequire full donor-recipient MHC compatibility in the human populationin which MHC (HLA) diversity is substantial. Moreover, such an approachwould be limited to suppression of clones that are autoreactive againsta single, recognizable peptide epitope in the context of defined MHC-II(HLA-DR). An additional significant advantage of the present inventionis that it overcomes the requirement that the pathogenic antigen beknown. Indeed, disease-associated antigens in a large number of humanautoimmune disorders, including human IBD, are not yet known and may bemultiple in number.

The “T-Body” Approach of the Present Invention

The “T-body” approach was designed by one of the present inventors andhis colleagues as a novel modality for specific redirection andactivation of effector T lymphocytes towards pre-defined targets, mostlythose associated with neoplastic processes (e.g., Pin thus JHU et al., JClin Invest 2004; 114:1774-81) and infectious diseases (Bitton N, etal., Curr Top Microbiol Immunol 2001; 260:271-300). The T-body approachwas intended to overcome the relative inaccessibility of antibodies tocertain sites (such as solid tumors) and the general ineffectiveness oftumor-infiltrating lymphocytes to combat solid tumors by combining intoone effector cell population the properties of the humoral and cellulararms of the immune system (Gross G et al., 1989; supra Eshhar Z, et al.,Br J Cancer Suppl, 1990; 10:27-9).

The preferred T-body chimeric receptors comprise a ligand bindingportion, preferably (1) a single chain antibody variable region (scFv)directed against a disease-associated antigen, linked to (2) an optionalextracellular spacer and a transmembrane region and (3) one or moreintracytoplasmic moieties of T cell costimulatory andstimulator/signaling molecules. Such CRs as initially developed enablenon-MHC restricted, specific antibody-type recognition, homing andpenetration of neoplastic tissues. Within the target tissues,antigen-specific activation of chimeric-receptor bearing T effectorcells enabled T cell-mediated destruction of tumor cells either bydirect cytotoxicity or by induction of a local inflammatory response.

While the scFv domain is the preferred recognition unit of the presentinvention, in other embodiments, it may be substituted by anotherstructure that serves as a targeting ligand (or ligand binding partner)that will facilitate bringing the Treg cells expressing the CR to aselected site or a selected antigen. Capon and colleagues have discloseda number of CRs of this sort, such as one where a ligand binding partnerpolypeptide is fused at its C-terminus to the N-terminus of animmunoglobulin constant region. See, for example, Roberts M R. et al.,Blood 1994; 84:2878-89; Ashkenazi A et al., Int Rev Immunol. 1993;10:219-27; Chamow S M et al., Int J Cancer Suppl. 1992; 7:69-72. Seealso U.S. Pat. Nos. 6,710,169; 6,407,221; 6,406,697; 6,319,494;6,117,655; 6,103,521; 6,077,947; 5,741,899; 5,714,147; 5,514,582;5,455,165; 5,428,130; 5,359,046; 5,336,603; 5,225,538; and 5,116,964.All of these documents are incorporated by reference in their entirety.

The CR polypeptide of the present invention is characterized broadly ascomprising (1) an extracellular portion or domain capable of binding toa ligand (such as a target antigen) in a non-MHC restricted manner, (2)an optional extracellular spacer and a transmembrane domain and (3) acytoplasmic region (one or more domains) capable of activating anintracellular signaling pathway.

Examples of preferred T cell CRs comprise a first binding domain, apreferred example of which is an extracellular scFv fragment derivedfrom a monoclonal antibody (mAb) specific for a selected antigen. Theforegoing domain is fused to a spacer domain (preferably a hinge domainof the Ig family that provides spacing and flexibility), a transmembranedomain, a costimulatory region, for example parts of a CD28 molecule,and a further intracellular signaling moiety for T-cells. Examples ofthe latter include a TCR/CD3 complex-associated ζ chain or η chain, oran ITAM-containing cytoplasmic region such as the γ chain of an Ig Fcreceptor (FcRγ). An ITAM is an “immunoreceptor tyrosine-based activationmotif; for reviews, see Humphrey M B et al., Immunol Rev. 2005 December;208:50-65; Pitcher L A et al., Trends Immunol. 2003; 24:554-60; IsakovN, Receptors Channels. 1998; 5:243-53; Daeron M, Annu Rev Immunol. 1997;15:203-34; Isakov N, J Leukoc Biol. 1997, 61:6-16; Cambier J C, JImmunol. 1995; 155:3281-5; Flaswinkel H et al., Semin Immunol. 1995;7:21-7, all of which are incorporated by reference in their entirety. Itis also possible to use the intracellular portions of TCR α, β, γ or δreceptor molecules in the CR for this purpose. The signaling moiety of acytokine receptor may also be present in the chimeric receptor chain foruse in the present invention. For example, adding the signaling portionof the IL-2 receptor will cause the Treg cell to further act as if ithad been subjected to external IL-2 upon binding of the extracellulartargeting domain to the selected target antigen or ligand. Furthermore,adding the signaling moiety of the TGFβ receptor will induce a Teffector cell to become a Treg cell and thus this may also be a usefuladdition to the chimeric receptor chain of the present invention. SuchCRs expressed on T-cells are known to be functional and, upon exposureto antigen, promote cytokine production (and, when expressed onappropriate effector cell type in the prior art, promoted lysis ofantigen-bearing target cells (Stancovski I, et al., J Immunol 1993;151:6577-82)).

An early configuration of a scFv-based CR comprised an extra-cellularrecognition domain and an intracellular signaling moiety. Fullactivation of such T-bodies through the CR required eitherpre-stimulation of the T-body or activation of a costimulatory pathwayby exposure to CD08/CD86 (B7)-bearing antigen presenting cells (APCs).

The creation of a tripartite chimeric receptor (TpCR) by one of thepresent inventors and by others (Pule M A, et al., Mol. Ther. 2005;12:933-41)), in which the signaling domain of the costimulatory CD28molecule was added to the cytoplasmic domain of the CR, enabledantigen-mediated activation of both the stimulatory and costimulatorysignaling pathways independent of B7-CD28 interactions (Eshhar et al.,2001, supra). This approach facilitates full activation ofscFv-expressing lymphocytes, resulting in improved effects (in the caseof T effector cells, improved anti-tumor; Pin thus JHU et al., supra).

Another useful intracellular signaling domain for the present inventionis all or part of the cytoplasmic domain of a phosphotyrosine kinase(e.g., a molecule of the Syk family) which is fused to the CR. See, forexample, Eshhar Z & Fitzer-Attas C J, Adv Drug Deliv Rev. 1998;31:171-82; Fitzer-Attas C J et al., J Immunol. 1998; 160:145-54.; andEshhar Z et al., Springer Semin Immunopathol. 1996; 18(2):199-209. Useof such a signaling moiety bypasses membrane-proximal signaling eventsthat are often defective in T-cells of subjects with acute or chronicinflammation or cancer.

Costimulatory Domains/Regions and Signals in the Tripartite ChimericReceptor

Retroviral-mediated expression of CRs in T-cells in general requires Tcell activation which activation is commonly achieved by combined use ofanti-CD3 and anti-CD28 antibodies. Such pre-activation was sufficient toprime the T-cells to respond to a signal mediated through the CR uponinteraction with the antigen for which the CR is specific—both in vitroand in vivo (e.g., Schwartz R H; Annu Rev Immunol 2003; 21:305-34). Acostimulatory signal is advantageous for optimal and sustained T cellfunction and antigen-driven re-activation, even by targets that oftenlack ligands for costimulatory molecules.

Antigen stimulation alone of CRs that lack a structure or mechanism forcostimulatory signaling is generally inadequate to activate resting ornaïve lymphocytes (Brocker T et al., J Exp Med 1995; 181:1653-9). Thus,in the absence of costimulatory signaling by CD28, resting T lymphocytestypically undergo anergy or apoptosis (Boussiotis V A et al., ImmunolRev 1996; 153:5-26). For further discussion of CD28 and its interactionswith B7, see also, L. Chen (ed.) The B7-CD28 Family Molecules, LandesBioscience, 2003, which is incorporated by reference in its entirety.

To overcome these limitations in the CR's used in the present invention,the first (recognition) domain, preferably an scFv domain, is linkedthrough an Ig hinge spacer and transmembrane segments to theintracellular segment of a costimulatory signaling molecule, preferablyCD28, and then to an intracellular activation region, such as from theCD3ζ chain or the FcRγ chain. Co-expression of two CRs, each with thesame scFv, the first linked to CD3ζ and the second to CD28, was found toprovide the requisite stimulatory and costimulatory signals for T cellactivation (Beecham E J et al., J Immunother 2000; 23:631-42).

Thus, in one preferred embodiment herein, an extracellular recognitionsite, preferably an antibody-based recognition site such as an scFv, islinked to a CD28 intracellular domain “in series” and further linked tothe intracellular signaling region of the TCR complex ζ chain. Such aconstruct was 20-fold more potent in stimulating IL-2 production uponexposure to solid phase antigen (compared with transfectants expressingCR's lacking the CD28 domain (Finney H M et al., J Immunol 1998;161:2791-7)). Intracellularly, this domain in the CR binds the p85subunit of phosphatidylinositol 3′-kinase.

One of the present inventors designed a novel tripartite CR composed ofa scFv recognition moiety fused to the non-ligand binding part of theextracellular domain (ECD) of CD28, the entire transmembrane andintracellular domains of CD28, and the intracellular stimulatory domainof FcRγ (“scFv-CD28-γ”) (Eshhar et al., 2001, supra). Human PBLtransduced with a nucleic acid construct encoding this CR werespecifically stimulated to produce IL-2. Activation was dependent onCD28 costimulatory activity.

The present inventors' laboratory has generated several lines of Tg miceexpressing CRs under control of T cell-specific regulatory sequences. Tlymphocytes from unprimed, naïve mice that are Tg for the scFv-CD28-γTpCR manifested potent responses (proliferation, IL-2 secretion, andrescue from apoptosis) upon stimulation solely by the cognate antigen inimmobilized form (Friedmann-Morvinski D et al., supra).

According to the present invention, molecules other than, or in additionto, CD28 are exploited to provide costimulatory signals when included inthe present CR configuration. Preferred examples of these are themembers of the “inducible co-stimulator” (ICOS) family, including OX40(CD134), CD40 ligand (CD40L, CD154), PD-1 (“programmed deathreceptor-1), and 4-1BB (CD137). Each of these ligand/receptor pairspossess distinct functions that differ according to the nature of thestimulus and the “antigenic history” of the T-cells on which they areexpressed. For example, CD28 signaling is accompanied by induction ofICOS, which, in turn, co-stimulates CD4+T cell activation. Theengagement of OX40 (studied in the context of tumor-specific adoptiveimmunotherapy) improved survival and anti-metastatic actions of Teffector cells by CD4+T helper cell-dependent mechanism (Weinberg A D,Trends Immunol 2002; 23:102-9). Activation of OX40 promotes expressionof anti-apoptotic proteins Bcl-XL and Bcl-2 and, accordingly, enhancesthe survival and hence the number of antigen-specific CD4+ T-cells,resulting in strong antigen-specific CD4+T cell memory. Engagement of4-1BB (CD137) costimulatory receptor with its ligand, 4-1BBL, increasedTCR-induced proliferation, survival, and cytokine production in bothCD4+ and CD8+T-cells (Cheuk A T et al., Cancer Gene Ther 2004;11:215-26). Cell survival was associated with increased expression ofthe anti-apoptotic genes bcl-XL and bfl-1. In general, the interactingligand/receptor pair 4-1BB/4-1BBL acts to amplify existing costimulatorysignals, particularly those emanating from CD28 (Guinn B A et al., JImmunol 1999; 162:5003-10). Human CD4+T-cells express PD-1 and itsligands, PD-L1 and PD-L2, upon activation. Antibodies to the receptorcan be agonists or antagonists of the apoptotic pathway. PD-1 engagementcan promote ICOS- or CD28-mediated costimulation. (e.g., Bennett F etal., J Immunol. 2003; 170:711-8.

The activity of costimulatory domains of CD28, ICOS, OX40 (CD134), and4-1BB (CD137) in CRs is also known in human CD4+ and CD8+T-cells (FinneyH M et al., J Immunol 2004; 172:104-13). In that study, the tripartitegenes were electroporated into cells to avoid pre-activation of thecells. When CR-bearing T-cells were stimulated by their specific antigen(CD33), cytokine release and cytotoxic activity were dramaticallyenhanced compared to cells in which the CRs lacked costimulatorysignaling structures. Inclusion of the 4-1BB signaling domain as thecostimulatory moiety in a TpCR on human T-cells with specificity againstthe CD19 antigen (anti-CD19-1BB-ζ) led to potent cytotoxicity againstCD19-bearing acute lymphoblastic leukemia target cells in vitro (Imai C,et al., 2004; 18:676-84).

While the present invention includes the use of an intracellular domainor part of any of these costimulatory sequences in the CR, it is notcertain that signaling evoked by these molecules has practicaladvantages over use of the CD28 costimulatory sequences alone. So, eventhough the performance of CD28 appears thus far to be quite satisfactoryboth in vitro and in vivo, the present invention includes within itsscope the use of additional or alternative costimulatory systems to CD28for generating Treg cells that perform optimally in suppressing Teffector cells and treating autoimmune/inflammatory and other conditionsas described herein. 4-1BB has been used successfully as an alternativeto CD28 in T-bodies. See Zhang et al., J. Immunol., 2007; 179:4910-4918.

Transfer of Redirected Tregs to Recipient Subjects

Use of transferred T-cells in vivo in adoptive therapy requires thattransferred cells survive, overcome the host's homeostatic controlmechanisms that may serve to hinder the acceptance of these cells, andmigrate to (home to, or traffic to) and accumulate or localize at, thedesired target site(s).

The immune system utilizes internal stimuli to regulate the total sizeof lymphocyte pools. The total number of peripheral T lymphocytesremains fairly constant, despite production of new cells, turnover ofexisting cells, and clonal expansion of antigen-specific cells during animmune response (Jameson S C. Nat Rev Immunol 2002; 2:547-56.). These“internal stimuli,” include cytokines and self-peptide-MHC ligands forthe TCR. At least two general mechanisms are believed to be responsiblefor homeostatic effects of bystander T-cells in limiting proliferation:(1) inhibition by physical T cell-T cell interactions; and/or (2)competition for limited “resources” (e.g., IL-7 and access to APCs withsuitable self-MHC ligands). The most prominent cytokines in this processare those that signal through receptors containing a common γ chain,termed collectively “γC cytokines.” These include IL-2, IL-4, IL-7,IL-9, IL-15, and IL-21. Homeostatic control of naïve T cell expansion(examined in vitro) is supported by IL-4, IL-7, IL15 and IL21 throughthe CD28 transmembrane region, whereas only IL-7 appears to be requiredin vivo (Jameson, supra).

Lymphodepletion or “lymphoablation” is preferably performed to conditiona recipient of the transduced Tregs of the present invention. Any methodknown in the art may be used, for example, irradiation, treatment withcertain antimetabolites such as fludarabine, etc. Such treatments havebeen used in conjunction with adoptive T cell therapy in other contexts.Lymphodepletion in vivo performed as a precursor to adoptive celltransfer is known to boost antitumor immunotherapeutic activity in miceand in humans (as studied particularly with autologous, tumor-reactive Teffector cells; Klebanoff C A et al., Trends Immunol 2005; 26:111-7). Inclinical trials, objective response rates of 50% were seen in patientswith solid metastatic tumors who had first been subjected tolymphodepletion. The mechanisms that are believed to underlie sucheffects include: the elimination of cellular cytokine ‘sinks’ forhomeostatic γC-cytokines (such as IL-7, IL-15 and possibly IL-21 (whichserve to activate and expand effector T-cells)), induction of tumor cellapoptosis and necrosis in conjunction with APC activation, and, mostimportant for the present invention, the impairment of CD4+CD25+ Tregcells that suppress T effector cells.

As noted, treatment with homeostatic cytokines may be used to maintainthe Treg populations in the recipient.

The present inventors' group found that activation of T-cells in generaland T-bodies in particular (such as that required during the ex vivomanipulations to express the CR with certain vectors) down regulatedexpression of the chemokine receptor CXCR4, thereby impairing T cellhoming in response to the chemokine SDF-1, for example. SDF-1 is achemoattractant for T-cells that express the CXCR4 (Bleul C C et al., JExp Med 1996; 184:1101-9; Beider K et al., Blood 2003; 102:1951-8).Using ErbB2-specific human T-bodies; these investigators showed thatthis homing is an essential step for the T effector cells to act invivo, measured as inhibition of advanced prostate cancer progression andeven cure (Pin thus et al., supra). Based on the foregoing knowledge,according to the present invention, redirected Treg cells must eitherhome/migrate to the desired target sites or be administered to suchsites.

Persistence of Responses of TpCR-Bearing T-Cells

A key factor for success of adoptively transferred T cell therapy (whichthus far has been examined most thoroughly with T effector cells incancer) is maintenance of the transduced T-cells' (effector) function.In one embodiment of the present invention, it is desired to maintainthe function of Tregs that have been administered to perform asuppressive function. In another embodiment, it may be preferred thatthe Tregs act in shorter “bursts” to curtail a more acute (rather than achronic) T effector response.

Because lymphocytes found in tumor patients include CD4+CD25+ Treg cellsthat suppress T effector cells (Wang H Y et al., Immunity 2004;20:107-18; Curiel T J, et al., Nat Med 2004; 10:942-9), such“endogenous” suppressive activity must be overcome to optimize theaction of redirected T effector cells. In the present invention, theobjective is the converse: redirected Treg cells are administered to asubject in need thereof to quell or otherwise inhibitimmune/inflammatory responses that characterize autoimmune conditions,transplant rejection, etc.

Examples of Clinical Trials Using Redirected T-Cells

While clinical trials using Tregs in accordance with the presentinvention have not yet been carried out, a number trials usingredirected, CR-bearing T effector cells are described below. Advantagemay be taken of various lessons learned in those trials in practicingthe present invention.

In a Phase I trial in HIV infected subjects, autologous lymphocytesbearing a CD4-ζ CR were administered (Mitsuyasu R T, et al., 2000, Blood96:785-93). Out of 24 patients, 11 also received concurrent IL-2infusions for 5 days. The treatment was well tolerated. In somepatients, a transient decrease of the viral load was observed in plasmaand rectal mucosa (the tissue reservoir for HIV). All subjects testednegative for replication-competent retrovirus (the delivery vector) forup to 1 year after infusion.

Cell Genesys, Inc. conducted phase I clinical trials in colorectalcancer patients using an anti-TAG72-ζ CR made from the humanized CC49mAb (Warren R et al., In: 7th International Conference on Gene Therapyof Cancer; 1998).

The group of Junghans tested 24 doses of CR-bearing lymphocytes theantigen-specificity of which was directed to CEA in colorectal patients.Up to 10¹¹ cells/patient were given. The treatment was adequatelytolerated (Junghans R et al., Proc Am Assoc Can Res, 2000, 41:543).

Hwu and co-workers at the National Cancer Institute conducted a phase Iclinical trial in ovarian cancer patients using T-bodies expressing a CRdirected against the MoV18-murine anti-folate-binding protein. Largedoses of the modified cells were infused into patients together withcontrolled administration of IL-2. No adverse side effects werereported. Neutralizing antibodies specific to murine MoV18 mAbdeterminants were found in the sera of several patients (Kershaw M H etal., Clin Canc Res, 2006; 12:6106-15.

A Phase I clinical trial in renal cell cancer (RCC) employed autologousG250-specific genetically modified T lymphocytes (Lamers C H J et al.,Daniel den Hoed Cancer News, 2004, 2:8-10). Infusions of these cellswere clinically well-tolerated. After 4-5 infusions, patients began todevelop liver enzyme abnormalities, a finding explained by thereactivity of the infused T-cells with G250L expressed on bile ductepithelium, albeit at low levels. Treatment was thus limited to only lowdoses of CR-expressing T-bodies. The results showed in any case that theredirected T-cells did exert CR-dictated functions in vivo.

Two other Phase I clinical trials have been initiated though theirresults have not yet been reported to the best of the inventors'knowledge. One Phase I trial treated neuroblastoma patients with PBLsand Epstein Barr virus-specific CTLs, both expressing GD-2 specificchimeric T cell receptors (Brenner M K. World wide web URLclinicaltrials.gov/ct/show/NCT00085930, 2005). The other trial employsgenetically modified CD20-specific CD8+CTLs for relapsed follicularlymphoma (Wang J, et al., Mol Ther 2004; 9:577-86

The present inventors recognize that certain events may interfere withthe efficacy of the therapy using Treg cells expressing CR's in vivo inhumans, for example:

-   -   (1) formation of neutralizing anti-idiotypic antibodies directed        to an idiotope of the scFv part of the CR that could reduced the        life-span or effectiveness of the Tregs;    -   (2) the low proportion of engineered cells that eventually        reached the targeted sites and;    -   (3) the potential damage to healthy tissue that expresses the        targeted antigen.

The use of Tregs according to the present invention has a much lowerrisk of (3). As described herein, direct administration of Tregs tosites of inflammation should overcome the limitation of (1)-(3).Adjustment of dose regimens (number of cells, frequency ofadministration) using routine clinical considerations are expected tolimit the impact of the above limiting factors.

According to the present invention, an effective amount of redirectedTreg cells are administered to a subject. Preferred carriers for theTreg cells are phosphate buffer, preferably 0.01-0.1M, more preferably0.05M, or 0.8% saline. Acceptable diluents or carriers for variousroutes of administration are well-known.

While individual needs vary, determination of optimal ranges ofeffective amounts of a given cell type for a particular disease orcondition is within the skill of the art. The dosage administered willbe dependent upon the age, health, and weight of the recipient, kind ofconcurrent treatment, if any, frequency of treatment, and the nature ofthe effect desired. Determination of the effective amounts can readilybe made empirically by those of ordinary skill in the art without undueexperimentation.

Typical dosages are between about 10⁶ and about 10¹¹ Treg cells perinjection or infusion, more preferably, about 10⁷ to about 10¹⁰ cells.If an antigen is to be administered with the cells (or separately, butto a site where it is intended to activate the cells), a dose of about0.01 to 100 mg/kg/body preferably, 0.1 to 50 mg/kg/body wt is preferred.

An effective amount of Treg cells is that needed to induce a measurablechange, generally a decrease, in the severity of any measurable symptomof the disease, preferably more than one symptom, and most preferably,would result in cessation of symptoms and cure of the disease orcondition. For example, without limiting the invention, the abovedecrease may be at least about 10%, more preferably at least about 20%,more preferably at least about 30%, even more preferably, at least about40%, and more preferably, at least about 50%, 60%, 70%, 80%, 90%, 95%,or 99%. It is within the skill of the clinical arts to determine whensuch therapeutic goals have been achieved, and to adjust the dose orfrequency of administration accordingly, or to cease further treatment.

The Treg cells of the invention may be given once, or on multipleoccasions, via a single or multiple routes. The cells may beadministered daily, or preferably on alternate days, preferably weeklyor biweekly. Administration can range over an interval of several daysto weeks, or even months or years. The frequency and duration ofadministration can be determined empirically, or based on the clinicalhistory and experience of the subject.

The cellular compositions of the present invention can be administeredby any of a number of means and routes known in the art. Administrationis preferably parenteral. Preferred routes include, intravenous,intramuscular, subcutaneous, intraperitoneal, intra-articular,intracerebroventricular, intraluminal (preferably into the lumen of theileum or colon), rectal or the topical route. Also included is the“intrathecal” route, which is intended to encompass injection, infusionor instillation directly into a cavity or space (thecum) surrounding anorgan or body region in which an undesired immune/inflammatory responseis occurring. Such spaces include the pleural space, peritoneum,subarachnoid space or dural space, or pericardial space. The genericterm for administration into a sheath encasing an organ is termed“intrathecal (see, for example, definition in Dorland's MedicalDictionary 29^(th) Edition, WB Saunders (2000) and Stedman's MedicalDictionary, 27^(th) Edition, Lippincott, Williams & Wilkins (2000)) asmeaning “within a sheath.” As used herein, this term is intended to bebroader than a more commonly used definition which is limited tointracranial spaces.

The compositions, methods, and products of this invention are applicableto human and veterinary uses. The preferred subject is a human.

Transgenic Mice Expressing TNP-Specific Chimeric Receptors

Several Tg strains of mice that express the TNP-specific TpCR, that wererecently produced by the present inventors and their colleagues(Friedmann-Morvinski D, 2005) are described herein. These mice are thesource of TNP-specific T effector and T regulatory cells and are used asexperimental animals in which the induction of colitis is evaluatedusing the ‘classical’ reactive hapten, TNBS. As a control for theseCR-bearing cells, cells from erbB-2-specific TpCR Tg mice that wereproduced in the present inventors' laboratory are used as they express aCR specific for an irrelevant antigen.

All mature T-cells and NK cells in these Tg mice express thescFv-CD28-FcRγ construct. Naive Tg T-cells can be fully activated byplastic-immobilized TNP without the need for pre-sensitization.(Friedmann-Morvinski D, et al., supra). Results in the Examples hereinshow that splenic CD4+CD25+ Tregs isolated from such mice specificallysuppress proliferation and cytokine secretion by TNP-specific effectorT-cells. Moreover, these Tregs are responsible for the delayeddevelopment and attenuation of TNBS-induced colitis in these animals. Ofimportance is the fact that the level of Tregs in the periphery of theTNP-specific TpCR-expressing strains is higher than in wild-type (WT)mice and that the Tregs do not require pre-activation to exhibit theirsuppressive activity in vivo. This is believed to result from thecross-reactivity of the SP6 mAb, from which the scFv of the TpCR wasderived.

Delivery of DNA Encoding the CR into T-Cells

Genetic modification of human peripheral T-cells is achieved in oneembodiment using retroviral vectors (Eshhar Z, et al., 2001, supra). Asa non-limiting example, the pBullet vector is used, into which theCR-encoding cDNA (Weijtens M E, et al., 1998) is introduced. Abicistronic expression construct is used in which the TpCR and eGFP cDNAare expressed under control of the LTR. This serves to generate apackaging cell based on PG13 that is being used to pseudotype theretroviral vector with the gibbon ape leukemia virus (GALV). Flowcytometric sorting is done on the basis of eGFP expression, andpackaging cells producing high-titer virions are selected to achievehigh transduction efficacy. To transduce human lymphocytes from healthydonors, lymphocytes are activated in culture with plate-bound anti-CD3and anti-CD28 mAbs (or using commercial microbeads coated with theseantibodies; e.g. from Invitrogen, Miltenyi Biotec, Inc.,) and aretransferred to plates coated with Retronectin™ (fibronectin fragmentCH-296) plates together with fresh supernatants taken from the packagingcells. At the end of the process that takes 5-8 days, cells arepropagated in the presence of IL-2 and then harvested and used.Following this ex vivo procedure, 45-70% of the cells are positive forCR (and GFP) expression.

Useful additional reagents are anti-idiotypic antibodies againstidiotopes of the scFv of the TpCR. These enable direct labeling andvisualization of TpCR on cell membranes. Such antibodies against the SP6scFv (exemplified below) have been made and used by the presentinventors.

An annotated nucleotide sequence (SEQ ID NO:1) and amino acid sequence(SEQ ID NO:2) of the TNP-specific TpCR used herein is shown in FIG. 27.The mature protein begins at amino acid residue 23 of SEQ ID NO:2.

A preferred sequence that excludes the scFv above, and that can belinked to any other appropriate ligand binding region, preferably adifferent scFv specific for another antigen, is that defined by theabove sequences beginning at the CD28 region. Thus, a preferrednucleotide coding sequence is nucleotides 2203-2523 of SEQ ID NO:1 andamino acids 260-367 of SEQ ID NO:2. Additional nucleotides comprising a5′ restriction site, and amino acids “inadvertently” encoded thereby,may also be included in a preferred sequence. Additional coding sequenceadded at the 3′ end of 2203-2523 of SEQ ID NO:1, or additional aminoacids encoded thereby and added to at the C-terminus of 260-367 of SEQID NO:2, may be present, provided that they permit the encoded sequence,as expressed on the redirected Tregs, to function as a TpCR in waysdescribed herein. Those skilled in the art of cloning and recombinantDNA technology will understand how to modify theses sequences to achievethe desired objective without undue experimentation.

Expression vectors comprising the foregoing sequences are also used inthe present invention, in the production of redirected, TpCR-expressingTregs.

Generation and Expression of TpCR and Foxp3-GFP Fusion Gene and itsExpression

Redirected T-cells are “converted” to Tregs by causing them to expressboth Foxp3 transcription factor and the antigen-specific TpCR. Suchmanipulation permits production of large numbers of Tregs for evaluationand therapeutic use. Successful co-transduction or co-expression istested by including a Foxp3-GFP fusion gene in the same construct as aTpCR to express both in the same cells. This approach is particularlyuseful when the starting cell populations are human PBL in which Tregsconstitute only about 3-5% of CD4+T-cells. This avoids the complicationsof another approach, also within the scope of the invention, in whichlarge scale Treg propagation is required for effective transduction withretroviral vectors. Moreover it will simplify the isolation of the Tregsand assessment of their fate in vivo.

In one non-limiting example, messenger RNA (mRNA) for Foxp3 is clonedfrom purified Tregs using PCR. Foxp3 cDNA is cloned into an eGFPClontech plasmid to create a Foxp3-GFP fusion protein. The fusionprotein is cloned into the pBullet vector containing TpCR inserted afteran IRES to create a bicistronic expression vector. Both a Foxp3-GFPsingle gene retroviral vector and a bicistronic TpCR—IRES−Foxp3-GFPdouble gene retroviral vector are transduced into isolated CD4+CD25−human peripheral blood T-cells following their activation with anti-CD3and anti-CD28 antibodies. The resulting cells are tested for expressionof the three genes by FACS using (1) antiidiotypic antibodies specificfor the scFv idiotype, or anti-hinge region antibodies and (2)intracellular GFP and Foxp3 by staining fixed cells with primaryantibodies specific for Foxp3 (Alexis Biochemicals, Lausanne,Switzerland).

In another embodiment, sequential expression protocols are used (firstTpCR and then Foxp3-GFP genes) or co-expression protocols. Once thegenes are expressed, relatively large number of Tregs can be obtainedand separated using cell sorter (FACSaria fluorescence-activated cellsorting (Becton Dickinson, Mountain View, Calif.), sorting for GFP andTpCR co-expressing cells.

The Foxp3 construct may be in the form of a bicistronic vector thatincludes DNA encoding a reporter molecule such as a fluorescent protein.Suitable reporter molecules are well-known in the art and includefluorescent, chemiluminescent or chromogenic proteins, for example Greenfluorescent protein (GFP) or enhanced yellow fluorescent protein (EYFP)or a fluorescent homologue thereof, firefly luciferase protein (encodedby the Luc gene) the enzymes chloramphenicol acetyl-transferase (CAT),or bacterial LacZ, (β-galactosidase) or the thymidine kinase gene(encoded by the HSV1 TK gene. GFP and EYFP are detected by fluorimetryor fluorescence histochemistry; enzymes are detected by use of achromogenic substrate that is converted into a colored product which canbe used in histochemical colorimetric detection of enzymatic activity.Luciferase is measured by activation of luciferin which emits light at aknown wavelength. Reporter molecules may be detected in vivo bynon-invasive detection techniques such as fluorescence optical imaging(FOI), bioluminescence optical imaging (BOI), cooled charged coupleddevice (CCD) camera optical imaging (CCOI) and positron emissiontomography (PET).

Infection of human CD4+CD25− T lymphocytes with retroviral vectorscarrying the Foxp3 gene was shown to convert these cells into ones witha Treg phenotype (Walker et al., 2005, supra; Wan et al., 2005, supra).

Any method for introducing DNA into a cell and expressing it may be usedin the present invention, including, but not limited to vectors such asretroviral or lentiviral vectors, electroporation, lipofection, and thelike.

The functionality of redirected Tregs can be determined using co-culturetests as described in the Examples. If APCs are to be used in suchtests, a preferred source is irradiated monocytes. The antigen is loadedinto irradiated human APCs which will present it to T effectors andTregs. In the case of antigens such as CEA, human colon carcinoma cellsstably transfected with the CEA epitope may be used. In such coculturetests, one may detect specific activation of TpCR-bearing Tregs throughthe TpCR. Treg activation is assessed by examining these cells' actionon T effector cell (1) proliferation and (2) cytokine secretion profile,focusing on IL2, IL4, IL10, IFN-γ and TGF-β (using commercial ELISAkits, e.g., Ready-Set Go ELISA kit, Ebioscience CA). It is preferred toassay TGF-β and/or IL-10 as an indication of the cells' Treg phenotype.

In a preferred embodiment, the present invention redirects Tregs tosites of colonic inflammation, by introducing into such cells CRs withantibody-type specificity. In sites of inflammation the redirected Tregsare activated to suppress IBD-associated immune response. Tregs endowedwith predefined specificity migrate and home to inflamed sites in thecolon where they undergo activation and, as a result, suppress Teffector cells that mediate the disease processes.

The present redirected Tregs represent a novel form of the ‘T-bodies’discussed above and are employed as a novel therapeutic modality in IBD.These T-bodies are T-cells that have been genetically engineered toexpress TpCR in which an antibody variable region is the recognitionunit linked to T-cell costimulatory and stimulatory domains that enablespecific activation of these T-cells but in a manner that is MHCindependent and not MHC-restricted. Based on previous studies usingtumor models described above, these redirected Tregs are tested inmurine models of IBD models.

An important aspect of this invention is the inventors' conception that,in the context of treating IBD, the colon-associated antigen(s) to whichthe T-bodies are redirected and targeted are not necessarily thepathogenic autoantigens recognized by the autoaggressive T effectorcells. Thus, this invention can exploit the phenomenon of “bystander”reactivity—where the presence of the relevant antigens at the sites ofthe inflammatory reactions serve to attract and “hold” or localize theredirected Tregs, permitting them to be activated and to exert theirsuppressive effects in a paracrine manner—acting on target effectorcells in the vicinity irrespective of differences in the T effectorcells' and Treg cells' antigen specificity.

CEA and LPS-Colonic Antigens as Targets for Redirected Human Tregs inIBD

Advantage was taken of a hapten-specific IBD model that is based onspecificity to the hapten TNP to study the suppressive effects of Tregs.In human disease, other antigens that are expressed in intestinal orcolonic tissue either normally or in the relevant disease state arepreferred targets. The include carcinoembryonic antigen, CEA, andbacterial floral antigens such as lipopolysaccharide, LPS.

Human IBD is idiopathic to the extent that pathogenic antigen(s) remainunknown. Lack of knowledge of the antigen would appear to be an obstacleto implementing the T-bodies clinically. Nevertheless, according to thepresent invention, there is no requirement that a pathogenic antigenmust also be the target antigen for Treg redirection and activation.Treg activation is indeed antigen-specific and thus depends on TCRs, orin the present Tregs, on antibody-based specificity, associated withcostimulation together with the activation/mediated by the intracellularsignaling moieties of the present constructs. However, once the Tregsare activated, their suppressive action is antigen-independent, and iscarried out by secretion of suppressive cytokines (e.g., TGF-β andIL-10) even after the activating antigen has been eliminated. Thus,inducing colonic Treg activation by any local colon-associated antigenwill promote potent Treg activation and proliferation, while the actionof these cells in inhibiting local inflammatory processes proceedsindependently of antigen. CEA is significantly over-expressed indiseased colon tissue in patients with active ulcerative colitiscompared to normal individuals and to patients with quiescent IBD(Smithson J E et al., J Pathol. 1996; 180:146-51; Pavelic Z P et al.,Anticancer Res. 1991; 11:1671-5). This enhanced tissue expression of CEAwas independent of dysplastic changes and is a result of the mucosalreaction to the inflammatory process itself. Thus, CEA is a preferredcandidate for Treg TpCR targeting in active ulcerative colitis.

A second candidate antigen (or “non-antigen” ligand) to which Tregs maybe redirected is endotoxin or LPS, derived from the outer membrane ofGram-negative bacteria resident in the colon. In one embodiment, theantibody-like part (scFv) the CR's extracellular recognition region maybe derived from an anti-LPS antibody, such as the mAb produced by thehybridoma with ATCC Accession No. HB9081. The nucleotide sequence of anscFv made from this mAb is shown as an annotation in FIG. 29 as part ofthe full sequence of a plasmid (pBullet) comprising this scFv—SEQ IDNO:3. Thus, a Treg expressing a TpCR that displays this scFvextracellularly will, at a site where LPS is present such as inflamedcolon tissue (whether the gut lumen, the lamina propria or even regionallymph nodes and other gut-associate lymphatic tissue) bind the LPS andbe activated to cause suppression of any T effectors cells in thevicinity in an antigen-nonspecific and MHC-independent manner.

Several types of non-antibody LPS receptors are known in the art. CD14(SEQ ID NO:4) is a class of LPS receptor that is a GPI-anchored 356 aaglycoprotein. It contains a 19aa signal peptide, an extracellular domainwhich contain 11 leucine-rich repeat (LRR) domains, 4 N-glycosylationsites and an unknown number of O-glycosylation sites. At least 2 solubleforms of CD14 have been described, one retains GPI and is released fromthe cell surface which results in an approximately 48 kDa molecule andthe other is released prior to the addition of the GPI anchor resultingin a higher molecular weight (>48 kDa).

While LPS interacts with CD14, CD14 is not capable of initiating atransmembrane activation signal because it is aglycosylphosphatidylinositol (GPI)-anchored protein. Thus, LPS mustinteract with a transmembrane receptor(s) that is responsible for signaltransduction. LPS is recognized by the toll-like receptor TLR4 and MD-2(SEQ ID NO:5; human), a molecule associated with the extracellulardomain of TLR4. CD14 greatly enhances the formation of LPS-TLR4-MD-2complexes, apparently by LPS loading onto TLR4-MD-2 but not in theinteraction itself between LPS and TLR4-MD-2. (Akashi S, et al., J. Exp.Med. 198:1035-42 (2003)).

Interaction of LPS with MD-2 in a TLR4-MD-2 complex triggers anintracellular signal transduction cascade that leads to the productionand release of proinflammatory cytokines, particularly TNF-α (DauphineeS M et al., 2006, Lab. Invest. 86, 9-22). Patients with IBD showincreased colon and serum levels of endotoxin, LBP, CD14, and MD-2(Pastor Rojo O, et al., 2006, Inflamm Bowel Dis., December 19 (epub);Amati L et al., Curr Pharm Des. 2003; 9:1937-45; Cario E et al., JImmunol. 2006; 176:4258-66). This change correlates with diseaseactivity, and proinflammatory cytokine levels return to normal aftertreatment.

A motif of human MD-2, for example, from amino acids 119-132 (14residues) of SEQ ID NO: ______ can substitute for MD-2 in MD-2-TLR4complex binding to the lipid A moiety of LPS, which (Mancek M et al.,Biochem Biophys Res Comm 2002; 292: 880-5; Kobayashi M et al., JImmunol. 2006; 176:6211-8).

Thus, in one preferred TpCR of the present invention, the extracellularrecognition region comprises, in place of an antibody-like structure(e.g., an scFv), a receptor that binds to a ligand that is not acting asan “antigen.” A preferred ligand in the present invention is LPS. Thus,the extracellular recognition region may comprise any of the followingreceptor structures:

-   -   (a) CD14 (SEQ ID NO:4),    -   (b) an LPS-binding motif of CD14, such as residues 100-119 of        SEQ ID NO:4,    -   (c) full length MD-2 (SEQ ID NO:5),    -   (d) an LPS-binding motif of MD-2 (residues 120-132 of SEQ ID        NO:5),    -   (e) a combination of a CD14 and MD-2 or    -   (f) a combination of a CD14-motif and an MD-2 motif (as is        encoded by the relevant segment of the chimeric nucleic acid of        SEQ ID NO:10.

Any of these constructs, when displayed on a Treg surface, will allowthe redirected Treg to bind to, and be activated by LPS molecules, forexample, at colon inflammatory sites, and thereby exert theirsuppressive activities in that vicinity. Again, this is an example ofreceptor-ligand binding/recognition that is not “antibody-like” butnevertheless permits the TpCR to act in accordance with this inventionand activate Tregs in an antigen-nonspecific (and MHC-independent)manner.

The present invention includes an embodiment in which redirected Tregsbearing a TpCR are designed to be specific for an antigen, referred toherein as “AgX,” that may have no inherent relationship with the tissuebeing targeted or the disease being treated. In this embodiment, theTregs specific for AgX are activated specifically in a selected site byadministering them together with AgX to that site. The site is one whereT effector cells are situated and active, where the ongoing inflammationis to be suppressed. The AgX-specific antibody-like receptor of theTregs will recognize AgX without a need for antigen presentation, MHC,etc., and the linked signaling moieties on the TpCR will serve toactivate the Tregs to release inhibitory cytokines at that site. Thisprocess will lead to nonspecific suppression of the ongoing T effectorcell and inflammatory activity.

The methods and compositions described herein are useful for any of anumber of autoimmune diseases which involve undesired effector T-cellsactivity as an underlying cause or as a consequence of thepathophysiology. Such diseases include, but are not limited to, IBD,rheumatoid arthritis, Type I diabetes, multiple sclerosis, autoimmunethyroiditis, autoimmune uveoretinitis, autoimmune orchitis, autoimmuneinsulitis, autoimmune oophoritis, psoriasis, autoimmune polymyositis andthe like. See, for example, Theofilopoulos, A., In: Stites, D P et al.,eds., Basic and Clinical Immunology, Lange Medical Publications, LosAltos, Calif., 1988)).

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

Example I Materials and Methods

The following materials and methods are used in various of the Examplesthat follow, as well as in carrying out certain embodiments of theinvention.

Cell Fractionation and Isolation

CD4+CD25+ Tregs were purified from splenic lymphocytes or peripheralblood mononuclear cell populations using several methods. One methodutilized magnetic bead separation (MACS). Spleens are mashed gently intoHBSS/5% FCS to prepare single cell suspensions.

CD4+T-cells were purified by negative selection by incubation withbiotin-conjugated CD4 MACS beads (Miltenyi Biotec, Inc., Auburn,Calif.). Further purification of CD4+CD25+ cells was conducted byincubation with phycoerythrin (PE)-conjugated anti-CD25 antibodies oranti-CD45RB^(high), followed by incubation with anti-PE microbeads(Miltenyi Biotec, Inc., Auburn, Calif.). Magnetic separation wasconducted using magnetic columns according to manufacturer'sinstructions. For highly-purified (>99%) Treg and effector T lymphocytesubpopulation, high-speed cell sorting is be applied, using BD FACSaria(®) cell-sorting system (BD Bioscience)

Lamina propria lymphocytes from colon were isolated as previouslydescribed (Han X et al., Gastroenterology. 2005; 129:185-203). Briefly,colonic mucosa was dissected, followed by incubation with Ca²⁺—Mg²⁺-freeHanks' balanced salt (HBSS) solution containing 1 mM dithiothreitol(Sigma-Aldrich, St. Louis, Mo.) for 30 min to remove mucus, and thenserially incubated twice times in medium containing 0.75 mM EDTA(Sigma-Aldrich) for 60 min at each incubation. The supernatants fromthese incubations containing epithelium and intraepithelial lymphocytepopulation are discarded, and the residual fragments pooled and treatedwith 2 mg/mL collagenase A (Worthington Biomedical, Freehold, N.J.) and0.01% DNase (Worthington) in humidified air at 37° C. for 2 hours. Thecells are then be pelleted twice through a 40% isotonic Percollsolution, after which they are purified further by Ficoll-Hypaquedensity gradient centrifugation (40%/75%).

In Vitro Induction of Tregs

Naturally-occurring Tregs are thymus derived, express high levels ofFoxp3 forkhead transcription factor and suppress activation of effectorlymphocytes. It has been discovered that antigen-specific activation ofhuman effector T-cells may induce expression of Foxp3 in a subgroup ofthe activated effector cells, which in turn develop a regulatoryphenotype. These induced regulatory T-cells were shown to be capable ofcell-contact-dependent suppression of freshly isolated effector cells(Walker et al., 2003, supra). In mice, prolonged exposure of effectorcells to TGF-β induces Tregs both in vitro and in vivo (Fantini et al.,J Immunol. 2004 and 2006, supra). This small, peripherally generatedpopulation of inducible Tregs may be central in regulation andcontainment of ongoing immune response, while the inability to inducesuch Tregs may be responsible for a propensity to develop autoimmunity.

To test whether such induction occurred after stimulation of T effectorcells through the TpCR, wildtype, TNP-Tg, Erbb2-Tg and TNP-CD28A-Tg Teffector cells were isolated by FACS sorting and cultured for 7 days inthe presence of either (1) anti CD3 Ab, (2) murine TGF-β, (3) mAb toTNP, (4) anti CD3 Ab+TGF-β, or (5) anti TNP Ab+TGF-β. Induction of Foxp3in cells “developing” from these effector T-cells was assessed afterseven days of culture using intracellular Foxp3 staining

Antigen-specific activation of human effector T-cells leads to inducibleexpression of Foxp3 in a subgroup of activated effector cells, which inturn develop regulatory phenotype. These induced regulatory T-cells arecapable of cell-contact-dependent suppression of freshly isolatedeffector cells. In mice, both in vitro and in vivo induction of Tregscan be achieved with prolonged exposure of effector cells to TGF-β(Fantini et al., 2004, 2006, supra). The present inventors adopted thistechnology to induce murine redirected Tregs from redirected effectorT-cells (see FIG. 3).

Animals

Several mouse strains were used in the studies described below and areused in various other embodiments of the invention. These includetransgenic mouse lines that specifically expresses anti-TNP or anti-ErbB2 TpCRs (bearing CD28-FcR

signaling chains) under the control of a CD2 promoter, as well as atransgenic mouse line expressing human CEA (Saha A et al., Immunology2006, 118:483-496)

All transgenic mice were back-crossed to Balb/c. Balb/c wild-type miceserve routinely as controls and recipients of adoptively transferredcells.

One cell-transfer colitis model is used in immune deficient Rag^(−/−)and SCID mice.

All invasive procedures were and are conducted under Ketamine andXylazine general aesthesia (127.5 and 4.5 mg/kg, respectively).Subcutaneous (S.C.) injections are conducted under local anesthesia with10% Xylocalne spray.

Colitis Induction and Assessment:

To induce TNP hapten-mediated colitis mice were sensitized with 15 μl ofthe haptenating agent 2,4,6-trinitrobenzenesulfonic acid (TNBS,Sigma-Aldrich) at a concentration of 2.5% v/v in 50% ethanol by skinpainting on day 1. On day 8, 150 μl of 1% TNBS in 50% ethanol wasadministered intrarectally via a 3.5 F catheter under generalanesthesia. OXA-induced colitis was induced by sensitizing mice withoxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one; Sigma-Aldrich)at a concentration of 3% v/v in 100% ethanol by skin painting on day 1,followed by intrarectal administration of 150 μl at a concentration of1% v/v in 50% ethanol on day 8.

In one preferred cell transfer colitis models, CD45RB^(high) (naïve)T-cells are transferred to immune deficient mice from syngeneicbackground (Powrie F et al., J Exp Med. 1994; 179:589-600. This model ofmucosal inflammation allows separating T effector and Treg cell functionwithin an inflammatory site.

In all models, colitis is assessed following induction using thefollowing parameters: degree of colon ulcerations, intestinal andperitoneal adhesions, wall thickness, and degree of mucosal edema. Eachparameter is graded on a scale from 0 (completely normal) to 4 (mostsevere) by two experienced, blinded observers. For histologicalevaluation of inflammation, distal colon tissue (last 10 cm) is removedand fixed in 10% formaldehyde. Five paraffin sections from each mouseare stained with hematoxylin-eosin using standard techniques. The degreeof inflammation is graded semiquantitatively on microscopic crosssections of the colon from 0 to 4 as follows: Grade 0: Normal with nosigns of inflammation; Grade 1: very low level of leukocyteinfiltration; Grade 2: Low level of leukocyte infiltration; and Grade 3:High level of infiltration with high vascular density, and bowel wallthickening; Grade 4: Transmural infiltrates with loss of goblet cells,high vascular density, wall thickening, and disruption of normal bowelarchitecture.

Murine Colonoscopy

For continuous monitoring of colitis pathology, a newly-developed, highresolution mouse video endoscopic system has been used Becker C et al.,Gut. 2005; 54:950-4. The experimental endoscopy system (from Karl Storz,Tuttlingen, Germany) consists of a miniature endoscope (1.9 mm outerdiameter), a xenon light source, a triple chip camera, and an air pump.Parameters for grading of colitis include bowel wall thickening,granularity, fecal consistency, fibrin deposition and vascular pattern.Whole colon methylene blue chromoendoscopy staining is used, whenappropriate, to visualize crypt pattern. A 3fr. Flexible biopsy forcepsis used for biopsy-taking. Biopsies are either placed in formalin forparaffin embedding, sectioning and subsequent immunohistochemistry,frozen in liquid nitrogen for cryosections, or obtained and used for RNAisolation. A typical yield of a biopsy specimen is approximately 2 μgRNA

In Vivo Imaging:

To follow migration (also referred to as homing or trafficking) ofredirected Tregs in mice, a whole body CCD camera (IVIS® 100 SeriesImaging System, Xenogen, Alameda Calif.). was used. Redirected Tregswere labeled with the near-infrared (NIR) lipophilic carbocyanine dye1,1′-dioctadecyl-3,3,3′, 3′-tetramethylindotricarbocyanine iodide (DiR,Invitrogen, USA). This dye has absorption and fluorescence maxima at 750and 782 nm, respectively, enables the safe direct labeling of membranesof human lymphoid cells with very low light absorption andautofluorescence levels in living tissues (Miller M J et al., Proc NatlAcad Sci USA, 2003; 100:2604-9; Kalchenko V et al., submitted forpublication, 2007). Additional in vivo visualization of Tregs labeledwith carboxy fluorescein diacetate succinimide ester (CFSE) at colonicmucosa was performed by intrarectal insertion of a 300 and 650 μmdiameter confocal microendoscope (Cell Vizio, MKT, Paris, France). Thisunique modality, previously untested in colitis models, allows repeatedin vivo assessment of homing of CFSE-labeled redirected Tregs to themost inner layers of colon tissue following induction of inflammation.

Determination of Colon Cytokine Levels

Colon mRNA expression of selected cytokines is determined to allowassessment of redirected Treg effects on local intestinal immuneresponse. in particular, levels of pro-inflammatory (TNFα and IFNγ) andanti-inflammatory cytokines (TGFβ and IL10), as well as levels of theTH₁ transcription factor Tbet and the TH₂ transcription factor GATA-3.Colon cytokine levels are assessed by measuring mRNA expression andprotein levels.

Samples for mRNA isolation are removed from colons of mice using in vivocolonoscopy or during sacrifice. Total RNA is isolated and processed andcDNA produced by RT-PCR. In all experiments, mice are divided into thefollowing groups: naive mice, colitis-induced mice, and colitis-inducedmice adoptively transferred with Tregs (naturally occurring, induced, orredirected, see detailed adoptive transfer experiments herein). Thefollowing sets of oligonucleotides and amplification conditions areused:

SEQ ID Amplification SEQUENCE NO: conditions TNF-α sense5′-AGTCCGGGCAGGTCTACTTT-3′ 15 60°/30 cycles antisense5′-GAGGCAACCTGACCACTCTC-3′ 16 IFN-γ sense 5′-TCTGGAGGAACTGGCAAAA-3′ 1763°/35 cycles antisense 5′-TGAGCTCATTGAATGCTTGG-3′ 18 TGF-β sense5′-TACAGGGCTTTCGATTCAGC-3′ 19 63°/35 cycles antisense5′-CGCACACAGCAGTTCTTCTC-3′ 20 IL-10 sense 5′-TCCTTGGGAAGCAATTGAAG-3′ 2163°/35 cycles antisense 5′-AACTGGCCACAGTTTTCAGG-3′ 22 T-bet sense5′-CTAAGCAAGGACGGCGAATGT-3′ 23 60°/35 cycles antisense5′-GGCTGGGAACAGGATACTGG-3′ 24 GATA-3′ sense 5′-GCCTGCGGACTCTACCATAA-3′25 54.8°/30 cycles antisense 5′-CAGGGATGACATGTGTCTGG-3′ 26 GAPDH sense5′-GTGTTCCTACCCCCAATGTG-3′ 27 60°/25 cycles Antisense5′-CTTGCTCAGTGTCCTTGCTG-3′ 28The relative mRNA expression compared to the housekeeping GAPDH isassessed using NIH image software and averaged from mice in each group.

IL-10 and IFN-γ protein expression levels in colon tissue are quantifiedby a cytofluorimetry-based ELISA system. In brief, whole proteins areisolated from colon specimens in the absence of detergent. Proteins (100μg) are immediately used for cytokine determination according tomanufacturer's instructions.

Foxp3 Immunohistochemistry of Colon Samples:

Foxp3 immunofluorescence is performed to estimate in situ the targetingof Treg to diseased colon, using TSA Cy3 and a fluorescence microscope(Olympus). In brief, cryosections are fixed in cold acetone for 10minutes, followed by sequential incubation with methanol, avidin/biotin(Vector Laboratories, CA), and protein blocking reagent to eliminatenonspecific background staining. Slides are then incubated overnightwith primary antibodies specific for Foxp3 (e.g., from AlexisBiochemicals, Lausanne, Switzerland). Subsequently, slides are incubatedfor 30 minutes at room temperature with biotinylated secondaryantibodies, and treated with streptavidin-horseradish peroxidase andstained with Tyramide (Cy3 or FITC). Before examination, nuclei arecounterstained with Hoechst 3342 (Molecular Probes, Ohio).

Example I Phenotypic Characterization of TNP-Specific Tregs

The inventors have produced transgenic (Tg) mice expressing aTNP-specific tripartite chimeric receptor (TpCR) that serve as a sourceof redirected Treg cells specific for the trinitrophenyl (TNP) hapten.This hapten has served as a “classical” antigen for years in studyingboth antibodies and T cell-mediated immunity. A chemically reactive formof this hapten, TNBS, is a contact sensitizing agent that induces andevokes delayed-type hypersensitivity (DTH) responses as well as inducingcolitis in animals, as described herein.

Generation of TNP-specific Tregs was achieved by the creation of Tg micethat express TNP-specific TpCR that comprises an scFv from theTNP-specific mAb Sp6 mAb linked to a truncated CD28 molecule which wasinserted between the scFv and the cytoplasmic part of the FcRγ chain(abbreviated as y herein (see FIG. 1). This construct includes the hingeregion, transmembrane region, and cytoplasmic region of CD28 but lacksthe B7 (ligand) binding site.

For the truncated form of CD28 (TpCR/CD28, FIG. 1) that does not includethe CD28 intracellular signaling domain, the inventors cloned the vectorat the same site. As a control, a Tg mouse expressing TpCR specific foranother, irrelevant antigen (Erb-B2) was used.

For expression of TpCR in T-cells of Tg mice, a construct comprising ananti-TNP (Sp6-derived scFv-CD28-γ was cloned into a human CD2promoter/enhancer minigene-based vector. Tg mice were generated at theWeizmann Institute's Department for Veterinary Resources by pronuclearmicroinjection of (BALB/c×C57BL/6)F₁ fertilized eggs derived fromhyperovulated donor females. Founder mice were screened by PCR of DNAfrom tail samples. Several founder strains were obtained that expresshigh level of the TpCR on their cell surfaces. These were backcrossedfor more than nine generations to either BALB/c or C57BL/6 mice toobtain MHC-homogeneous mice.

The studies below describe the characterization of various Tregsubpopulations in the different strains of TNP-CR transgenic mice, andthe expression of TpCR on these Tregs.

Example II Isolation of Trees in which TNP-Specific TpCR are HighlyExpressed

Tregs were isolated using double magnetic bead separation (MiltenyiBiotech) or by fluorescent cell sorting in which fluorescently labeledCD4+CD25+ cells were sorted using the FACSARIA cell sorting system.

Treg expression of TNP-specific TpCR was assessed by containing cellsfor Foxp3 (considered the “gold standard” marker of Tregs) andPE-labeled mAb specific for TNP antibody (generated in the inventors'laboratory). Controls included groups stained with the appropriateisotype controls. As is shown in FIG. 2, Tregs from TNP-Tg mice, but notfrom wild-type mice, expressed high levels of TNP-specific TpCR.

Example III TNP-Tg Mice Posses Increased Numbers of Foxp3+TreePopulation

Peripheral lymphocytes from the spleen as well as gut-associatedlymphocytes from the lamina propria of the colon were stained. As shownin FIG. 3, a CD4+CD25+ cell population (represented as the ratio ofCD4+CD25+ cells among CD4+T-cells) was elevated modestly in TNP-Tg micein comparison to control mice (wildtype, ErbB2-Tg and TNP-CD28 null-Tgmice). In contrast, higher numbers of Foxp3+ cells were observed inTNP-Tg animals compared to the control animals in comparison to allother mouse types (FIG. 4).

To resolve what may have appeared to be an inconsistency between thehighly elevated Foxp3+Treg population in TNP-Tg mice and the modestlyelevated CD4+CD25+ Treg population in these mice, effector CD4+CD25−cells were isolated by cell sorting to a level of 99% purity. Isolatedcells were stained for Foxp3 (FIG. 5). As expected, no positive Foxp3staining was noted in T effector cells from wildtype, ErbB2-Tg andTNP-CD28null-Tg mice. In contrast, TNP-Tg T effector cells featured asignificant population of Foxp3+ cells. This observation was furthervalidated in whole spleen cell populations that were co-staining forFoxp3 and CD25 (FIG. 6). The presence of a significantly greaterFoxp3+CD25− Treg population in TNP-Tg mice is supported by other recentresults by the inventors' laboratory showing that the Sp6 mAb from whichthe scFv of the TNP-specific TpCR was derived recognizes cross-reactiveendogenous thymic antigens. This results in either deletion or earlyrelease from the thymus to the periphery before several other immature Tcell subsets, including immature CD25− Tregs.

Example IV Induction of TNBS Colitis in TNP-Tg Mice Significant Elevatedthe Numbers of Foxp3+ Expressing Cells in Peripheral and Colon-DerivedLymphocyte Populations

Induction of TNBS colitis results in further elevation in splenic (FIG.7) and colon (FIG. 8) Foxp3+Tregs in TNP-Tg (FIGS. 7 & 8, respectively).These results demonstrated that TNP-specific Treg expansion occurredfollowing induction of colitis in Tg mice, reflecting Treg proliferationfollowing antigen-specific activation by TNP.

Example V In Vitro Functional Characterization of Redirected Tregs

A key prerequisite for the utility of Tregs expressing TNP-specific TpCRin the treatment of autoimmunity is verification of their regulatoryactivity, namely an ability to suppress T effector cell proliferation ina dose-dependent manner. Also examined was whether such Treg activationoccurs as a result of TpCR signaling, and whether it was indeedindependent of CD28-B7 interaction. A series of coculture experimentsexamined Tregs from the different Tg strains, as is outlined below.

Example VI Tregs Bearing the TNP-Specific Chimeric Receptor SpecificallySuppressed the Activity of T Effector Cells

To characterize whether TNP-Tg Tregs retained their anergic properties,CD4+CD25+ Treg cells and CD4+CD25− T effector cells from different Tgmouse founders (anti-TNP, anti-Erb-b2 control and wildtype (WT) mice)were purified from bulk splenocytes. 10⁵ cells were incubated in vitrofor 24 h, 48 h or 72 hrs (FIG. 9) and activated non-specifically withanti CD3 and anti-CD28 Abs, or specifically with Fowl gammaglobulin-modified TNP (FyG-TNP). T cell proliferation was measured usingeither the uptake of a dye (tetrazolium salt XTT) or radiolabeledThymidine. IL2 secretion was measured using XTT staining of theIL-2-dependent CTLL-2 cell line.

All effector cell populations showed significantly increasedproliferation and IL2 secretion following non-specific stimulation withanti-CD3+ anti-CD28 Abs. Specific stimulation by FyG-TNP resulted inproliferation and IL2 secretion by T effector cells bearing TNP-chimericreceptor, but not by such T-cells from WT or anti-Erb-b2 Tg mice. Incontrast, Tregs from wildtype mice, TNP-chimeric receptor Tg mice andErb-b2 Tg mice retained their anergic properties: they did not undergomeasurable proliferation or IL2 secretion when subjected to thenon-specific stimulus or specific Ag.

To characterize whether polyclonal activation could trigger thesuppressive action of TNP-Tg Tregs, these Tregs were cocultured in96-well microplates (0.2 ml) with irradiated antigen presenting cells(APCs) and T effector cells (CD4+CD25−) at 1:1 ratios. Cells in theseculture were activated either by (1) immobilized antigen “mimic”(anti-CD3+ anti-CD28) or (2) soluble Concanavalin A (ConA). T cellproliferation was measured as Thymidine uptake and IL2 secretion wasmeasured as growth of cells of the IL-2-dependent CTLL-2 cell line (XTTstaining).

FIG. 10 shows a ConA experiment. Non-specific (polyclonal) stimulationof Tregs induced these cells to exhibit potent inhibition of T effectorcell proliferation and IL2 secretion, irrespective of the origin of theTregs or the presence of the chimeric receptor. Thus, geneticmanipulation of Tregs of the type described here preserves theirsuppressive properties.

Example VII Antigen-Specific Stimulation of Redirected Tregs Cells withTNP Results in Suppression of T Effector Cell Proliferation

To study the antigen-specific Treg stimulation through the TpCR,coculture experiments were done in which TNP-loaded APCs provided the Agpresentation (FIG. 11). Comparisons of TNP-specific Treg stimulation wasperformed, comparing wildtype vs. TNP-Tg Tregs (FIG. 11, left panel) orErbB2-Tg and TNP-Tg Tregs (FIG. 11, right panel). In the absence of TNPstimulation, T effector cell proliferation did not occur (left-most barsin both graphs). In contrast, incubation with TNP-modified APC'sresulted in:

(1) marked proliferation of TNP-Tg but not of wildtype or ErbB2-Tgeffector T-cells in the absence of Tregs; and(2) activation of TNP-Tg, but not of WT or Erb-b2-Tg Tregs, manifest assuppression of effector cell proliferation by TNP-specific Tregs only.

These results proved the antigen-specific manner of activation andfunction of TNP specific TpCR Tregs cells in response to the antigen,TNP.

Co-culture of varying ratios of TNP-specific Tregs and TNP-specificeffector T-cells (FIG. 12) demonstrated successful antigen-specificinhibition by Tregs at a ratio of 1 Treg to 8 T effector cells.

Studies supporting the existence of the bystander effects were carriedout. Colitis was induced in mice as above using OXA as described inExample I. Adoptive transfer of TNP-specific Tregs alone did not protectthese animals from colitis. However, in the presence of trace amounts ofTNP applied to the colon, animals were protected from this OXA-inducedcolitis.

Example VIII Suppressive Activity of TpCR-Redirected Tregs isIndependent of Costimulatory Receptors

To assess the role of costimulatory signaling in the above TpCR-Tgmodel, coculture experiments as above were performed using as APC's (a)TNP-modified P815 cells, a cell line that does not express B7, or (b)TNP-loaded genetically modified P815 cells stably expressing the B7 gene(FIG. 13). Stimulation of TNP-Tg effector T-cells with TNP-P815 cellsinduced proliferation, which was markedly suppressed by TNP-Tg Tregs.Expression of B7 on these APC's did not promote any furtherTreg-mediated suppression. It was concluded that maximal Tregsuppression occurred independently of B7. Some suppression was alsonoted with wildtype Tregs. This was explained by the pre-activation ofthese cells prior to their harvesting. Based on these results, it couldbe concluded that inclusion of the intra-cytoplasmic signaling domain ofCD28 in the TpCR of redirected Treg cells results in full activation oftheir suppressive activity when stimulated by Ag irrespective of thepresence of B7-CD28 costimulation.

Example IX Functional Characterization of TNP-Specific Treg Activity InVivo in Murine Colitis

TNBS is a potent inducer of T-cell responses such as DTH/contactsensitization. This reactive hapten also induces autoimmune colitis whenapplied to the colon of pre-sensitized mice. To determine whetherTpCR-bearing Tregs could suppress autoimmunity, the acute TNBS-mediatedcolitis model was employed. Intra-rectal administration of TNBS leads toits binding to colon proteins, rendering these modified proteinsimmunogenic so that they elicited a T cell mediated immune response. Thesuppressive effect of endogenous or exogenously transferred Tregs onautoimmune inflammatory disease was tested in this model. A differenthapten, oxazolone (OXA) with similar sensitizing properties and whichinduces experimental colitis was used as a specificity control in vivo.

Example X Transgenic Mice Whose Entire Treg Population Expresses theChimeric Anti-TNP Receptor are Resistant to TNBS-Induced Colitis

TNP hapten-mediated colitis was induced in Tg and WT mice by firstsensitizing the animals with 150 μl of the 2,4,6-trinitrobenzenesulfonicacid (TNBS, Sigma-Aldrich) at a concentration of 2.5% in 50% ethanolpainted on the skin on day 1. On day 8, the antigen was administeredrectally (150 μl of 1% TNBS in 50% ethanol; high dose colitis). WT micedeveloped severe colitis within 2-5 days of rectal TNBS administration(FIG. 14, left panel). In contrast, 90% of the TNP-Tg mice had normallooking colons (FIG. 14, right). Colitis severity scores were asfollows:

Colitis score Animals (Arbitrary Units) Mortality Wildtype   12 ± 3.1 90± 20% TNP-ΔCD28-Tg 11.1 ± 4   ErbB2-Tg 12.7 ± 3.2 TNP-Tg 2 ± 2 (p <0.05) 20 ± 20% (p < 0.01)

To produce mortality curves the above experiments were repeated withlower doses of TNBS (75 μl of 1% TNBS in 50% ethanol). Similardifferences in colitis severity and in mortality were noted (FIG. 15).Microscopically, colons of wildtype, TNP-ΔCD28-Tg and ErbB2-Tg miceshowed severe inflammation, necrosis, hemorrhage and in some casesperforation, while those of TNP-Tg mice appeared normal or near normal(FIG. 16).

Evidence of antigen specificity of the protection from hapten-mediatedcolitis came from studies of OXA-induced colitis. As shown in FIG. 17,no differences in mortality were noted between wildtype, TNP-Tg,TNP-ΔCD28-Tg and ErbB2-Tg mice. The same was true for macroscopic andmicroscopic colitis scores. From these in vivo experiments, it wasconcluded that the presence of a Treg cell population that uniformlyexpresses the anti-TNP chimeric receptor results in high gradeprotection against TNBS-induced inflammation, manifest as reduced coloninflammation and significantly improved survival. It is noteworthy thatinclusion of CD28 costimulatory signaling in the CR significantlyenhances TNP-Tg Treg suppressive function.

Example XI Prolonged Stimulation with TNP Combined with TGF-β PromotesConversion of Leads to TNP-Specific Effector T-Cells to TNP-SpecificTrees

Naturally-occurring Tregs are thymus derived, express high levels ofFoxp3 and suppress activation of effector lymphocytes. Antigen-specificactivation of human effector T-cells may induce expression of Foxp3 in asubgroup of the activated effector cells, which in turn develop aregulatory phenotype. These induced regulatory T-cells were shown to becapable of cell-contact-dependent suppression of freshly isolatedeffector cells (Walker et al., 2003, supra). In mice, it has beendemonstrated that prolonged exposure of effector cells to TGF-β inducesTregs both in vitro and in vivo (Fantini et al., 2004, 2006, supra).This small, peripherally generated population of inducible Tregs may becentral in regulation and containment of ongoing immune response, whilethe inability to induce such Tregs may be responsible for a propensityto develop autoimmunity.

To test whether such induction occurred after stimulation of T effectorcells through the TpCR, wildtype, TNP-Tg, Erbb2-Tg and TNP-CD28null-Tg Teffector cells were isolated by FACS sorting and cultured for 7 days inthe presence of either (1) anti CD3 Ab, (2) murine TGFβ, (3_mAb to TNP,(4) anti-CD3 Ab+TGF-β, or (5) anti-TNP Ab+TGF-β. Induction of Foxp3 incells “developing” from these effector T-cells was assessed after sevendays of culture using intracellular Foxp3 staining (FIG. 18).

At time 0 to the time of T effector cell sorting, no Foxp3 staining wasnoted. A week of stimulation with anti-CD3+TGF-β, but not withTNP+TGF-β, resulted in a 2-fold increase in Foxp3+ cells in wildtype,ErbB2-tg and TNP-CD28null-Tg T effector cells. In contrast, a dramatic30-fold increase in Foxp3+ cells was observed in TNP-Tg effector cellsfollowing exposure to TNP+TGF-β. Interestingly, no Foxp3 induction wasnoted in TNP-Tg T effector cells following incubation with anti CD3 Abor with TGF-β, probably due to significant attenuation of CD3 expressionin TNP-Tg T-cells (Morvinsky-Friedman et al, in press).

The foregoing results demonstrated that antigen-specific stimulationthrough the TpCR in the presence of TGF-β, led to induction ofAg-specific Tregs from T effector cells, further contributing to Tregexpansion. This change was dependent both on the antigen-specificity ofthe Ab recognition unit of the TpCR and the intra-cytoplasmic CD28signaling moiety.

According to the present invention, induction of Tregs in this mannerpermits the generation of large populations of TpCR-bearing Tregs thatcan be used in cell-based therapy of autoimmunity.

Example XII Adoptive Transfer of TNP-Tg Tregs to WT Mice with TNBSColitis Ameliorates Symptoms and Improved Survival

Studies were conducted to establish that TNP-Tg Tregs are responsiblefor the resistance of TNP-Tg mice to TNBS colitis and to evaluate theirtherapeutic capacity in autoimmunity. Wildtype, TNP-Tg and Erb-b2-TgTregs were isolated and administered in varying numbers to wildtype micea day after induction of TNBS colitis. As was previously described,adoptive transfer of large numbers of Tregs of any origin (>2×10⁵)caused nonspecific attenuation of TNBS colitis. This is believed toresult from the presence of a sufficiently large population ofpre-activated Tregs that can exert their suppressive activity in theabsence of antigen stimulation or specificity. In contrast, adoptivetransfer of smaller numbers (5×10⁴) of TNP-Tg Tregs but not of wildtypeor Erbb2-Tg Tregs, prolonged survival (FIG. 19), improved Wallachcolitis severity scores (FIG. 20), and significantly improvedmacroscopic (FIG. 21) and microscopic (FIG. 22) appearance of colontissue. FIG. 21 shows marked bowel shortening, a manifestation of coloninflammation (in WT and ErbB2-Tg, but not in TNP-Tg mice). FIG. 22 showsthe severe transmural inflammation, necrosis, mucosal bleeding and lossof normal architecture in colons of WT mice with TNBS colitis that hadreceived control (WT and ErbB2-Tg), but not in TNP-Tg colons.

Example XIII Migration/Trafficking of Redirected ‘Tregs to Sites ofInflammation: Adoptively-Transferred TNP-TG Tregs Localize to Colons inTNBS Induced Colitis

Studies were done to garner additional support for the role of TNP-TgTregs in attenuating TNBS colitis by showing that these cells indeedlocalize to inflamed colon tissue. WT and TNP-Tg Tregs were isolated andstained with the fluorescent intracellular dye, carboxyfluoresceindiacetate succinimidyl ester (CFSE). Following staining, 10⁶ Tregs wereadministered intraperitoneally (ip) to control WT mice or to WT mice inwhich TNBS colitis had been induced 12 hours earlier. Sixteen hoursafter this treatment, mice were sacrificed and lamina proprialymphocytes isolated from their colons. The protocol used was describedabove to isolate lamina propria lymphocytes. Thereafter, the cells wereexamined for the presence of CFSE-positive cells by FACS analysis. Asshown in FIG. 23, very small numbers of adoptively-transferred WT orTNP-Tg Tregs reached the colons of normal mice. Induction of colitis ledto a small increase (0.5% to 0.8%) in the number of WT CFSE-stainedTregs in colon tissue. In contrast, adoptive transfer of CFSE-labeledTNP-Tg Tregs to mice with colitis led to a significant increase in colonTreg population, ranging from 0.4% to 3.6%. This demonstrates thatTNP-Tg Tregs localize in a TNBS-exposed target organ, where they exerttheir suppressive function.

Example XIV Accumulation of Adoptively Transferred TNP-Tg Tregs withinthe Mucosal Layer of Colons of Mice with TNBS Colitis

An important aspect of understanding the role of Tregs for adoptivetherapy of autoimmune inflammation of the Treg in diseased organs, wherethey are expected to exert their suppressive effects. To demonstrateTNP-Tg-Treg localization, WT and TNP-Tg Tregs were labeled with CFSE andtransferred to WT mice 24 hours following induction of TNBS colitis.While very small numbers of CFSE-labeled WT Tregs were observed in cellextracted from colonic lamina propria of naïve or TNBS colitis-inducedmice, a nine-fold increase in TNP-Tg Tregs was noted (FIG. 23).

To study the kinetics of TNP-Tg Treg localization of in the livinganimal, the IVIS® 100 Series Imaging System was employed (Xenogen,Alameda Calif.). Wildtype and TNP-Tg Tregs, 1.5×10⁶, labeled with thenear-infrared lipophilic carbocyanine dye1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR,Invitrogen USA), were administered ip to WT mice with or without TNBScolitis, who were monitored daily with the IVIS whole body CCCD camera(FIG. 24). A strong anterior-abdominal fluorescent signal, reflectingthe bulk of injected Tregs, was noted in naïve mice 24-48 hoursfollowing ip injection of Tregs and disappeared thereafter due to Tregredistribution. In mice with TNBS colitis, a faint abdominal signal wasnoted for 72 hours, probably reflecting inflammation-relatedauto-fluorescence. In mice with TNBS colitis that received labeled WTTregs, a week-moderate abdominal fluorescent signal could be recognizedup to 96 hours following Treg transfer. In contrast, TNP-TgTreg-administered to WT mice with TNBS colitis featured a distinctabdominal fluorescent signal for up to a week following cell transfer,substantially stronger than that of wildtype Tregs at all time points.These results reflect persistent TNP-Tg Treg localization within colonsduring the colitis.

To determine whether TNP-Tg Tregs reach the inner colonic mucosal layer,the location where most of TNBS-induced mucosal damage takes place, theCell Vizio confocal microendo-scopy system was employed (Cell Vizio,MKT, Paris, France). An intrarectally-inserted 650 μm diameter confocalmicroendoscope enabled visualization of CFSE-labeled cells in up to a150 μm bowel wall thickness (FIG. 25). Numerous adoptively transferredCFSE-labeled TNP-Tg, but no WT Tregs could be visualized in the innermucosal layer of WT mice with TNBS colitis as early as 12 hoursfollowing systemic Treg transfer. This result indicates that TNP-TgTregs localize in response to colonic TNBS, within hours of theiradministration, and that they reach the deepest colonic mucosal layers,where they exert their suppressive functions.

Example XV Administration of TNP-Tg Tregs Specific to a BystanderAntigen (TNBS) Cures Colitis Mediated by a Pathogenic (Oxazolone)Antigen

In contrast to hapten-mediated colitis, in which the eliciting antigenis predefined, the disease-causing antigen in inflammatory bowel disease(IBD) is unknown. To enable application of the ‘T-body’ approach in IBD,naïve TPCR-bearing Tregs were tested to determine whether they can beactivated by a predetermined ‘bystander’ colon- or colitis-associatedantigen, to perform their antigen-nonspecific suppressive action. Tothis end, WT and TNP-Tg mice were pre-sensitized to oxazolone only. Amixture of oxazolone and low doses of TNBS were introducedintrarectally. As is shown in FIG. 26 a, concomitant challenge of WTmice with TNBS and oxazolone, was associated with a 100% one-weekmortality rate, as compared to only a 15% one-week mortality of TNP-Tgmice (P<0.01). Similarly, significant mucosal inflammation was evidentin both WT and TNP-Tg mice with oxazolone colitis (not shown), and wasmost severe in wild-type mice given TNBS+oxazolone (FIG. 26 b, box I)resulting in severe bleeding, fibrin deposition and sloughing off ofcolonic mucosa.

In utter contrast, TNP-Tg mice administered TNBS+oxazolone featurednormal-appearing colonic mucosa with scattered areas of mild colitis(FIG. 26 b, box II). Macroscopically and microscopically, colons ofconcomitantly TNBS-treated and oxazolone-treated WT mice featured severecolitis, as opposed to the near-normal colons in TNP-Tg mice (FIGS. 26 cand 26 d, respectively.)

Notably, this “bystander” protective effect also occurred when TNP-TgTregs were adoptively transferred to oxazolone-presensitized wild-typemice which were intrarectally boosted with a mixture of oxazolone andlow doses of TNBS (FIG. 26 e, P<0.01)). In contrast, adoptivelytransferred WT Tregs did not have this curative effect, and the very lowTNBS doses in the absence of pre-sensitization were insufficient bythemselves to induce TNBS colitis. These results demonstrate that Tregactivation by a bystander antigen (TNBS) cause an improvement in colitisthat has been induced by a different non-cross-reactive antigen(oxazolone).

Example XVI Delivery of Foxp3 to Cell Nucleus by Vectors ComprisingChimeric Receptors

An experiment was conducted to verify that the Foxp3 can be expressedfollowing transduction of A273 cells with retroviral vector constructsdesigned to transduce Treg cells. A fused gene was generated thatincluded eGFP sequences encoding green fluorescent protein (referred toas eGFP or GFP). This was engineered as a bicistronic construct with theGFP sequence alone or linked with a Foxp3-encoding sequence (after anIRES) into vectors that comprised a chimeric receptor construct with thefollowing extracellular recognition regions: See description of FIG. 27for discussion of the chimeric receptor constructs used. 273 cells.transduced with vectors comprising the same chimeric receptors but witha bicistronic eGFP gene only (without Foxp3) served as controls forFoxp2 expression.

The results are shown in FIG. 27. The upper half of the Figure shows theGFP-only controls, whereas the lower half of the Figure shows GFP-Foxp3constructs. The two-paneled rectangles in the Figure show lightmicroscopic (left half) and fluorescence microscopic (right half) imagesof the same material (to visualize and localize the GFP).

All the control group expressed the eGFP in their cytoplasm only. Incontrast, in cells that were transduced with the eGfP-Foxp3-fusionconstructs, the nuclei were fluorescent (appearing as bright nuclearimages) due to the transport to and expression of the Foxp3transcription factor in the nuclei.

In another experiment not shown here, expression of chimeric receptormade of the full length MD2 protein (SEQ ID NO:5) or the CD14 protein(SEQ ID NO:4) was confirmed by the ability of transduced cells, whichexpressed the extracellular region of the CR on their surface, to bindthe ligand of MD2 and CD14, bacterial LPS, which was provided inbiotinylated form and revealed by secondary binding of fluorescentavidin.

Example XVII Vectors Comprising Chimeric Receptors with LPS-BindingExtracellular Regions

Nucleic acid constructs and vectors that encode extracellular regionsthat comprise an anti-LPS antibody domain (e.g., SEQ ID NO:3 or thescFv-coding portion thereof) have been made and others can be made. Suchvectors express extracellular polypeptide domains that are shown to bindLPS, for example in an assay using biotinylated LPS and detectablylabeled (e.g., fluorescently labeled) avidin. See also Example XVIabove.

Nucleic acid constructs and vectors that encode extracellular regionsthat comprise a LPS-binding nonantibody polypeptide have been made(e.g., SEQ ID NO:6-11, 13 and 14). Such constructs include bicistronicones that also comprise Foxp3. Other such constructs can be made usingthe method described above along with methods well-known in the art.Such constructs (such as SEQ ID NO: 13 and 14) include those encodingfull length CD14 (SEQ ID NO:4) or MD2 (SEQ ID NO:5) protein, andconstructs encoding LPS-binding motifs therefrom (such as SEQ ID NO:6-9)and combinations (such as SEQ ID NO:10 and 11). The constructs that aremade include those with CD28-FcRγ intracellularstimulatory/costimulatory regions and those that utilize others of thetype disclosed herein.

Treg cells are redirected as described herein using the aboveconstructs, including those that have been made and tested and thosethat can be made.

Such Treg cells are administered into subjects suffering from IBD, suchas ulcerative colitis. Treg cells are administered in numbers inaccordance with the above examples, or in numbers that are readilydetermined to be effective by those skilled in the art using onlyroutine experimentation, and via routes of administration as exemplifiedabove and disclosed throughout this document. These redirected Tregcells that express an LPS binding antibody region or another LPS-bindingmoiety on their surface (CD14, MD2, fragments thereof, or combinationsof these) as part of their CR's are able to reduce the symptoms,intensity, severity and duration of the IBD in the subject to asignificant degree compared to untreated control subjects or controlsubjects administered with control Tregs. (Such control Treg cells areones not transduced to express the present CR's, or those redirected tobe specific to antigens or ligands not present at the site of the IBD.)Introduction of LPS-related molecules or epitopes that bind to thesesame extracellular receptors on the redirected Treg cells to the sitesof administration (and/or expected action) of the redirected Treg cellsfurther facilitates their therapeutic activity.

The references cited above are all incorporated by reference herein,whether specifically incorporated or not.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

1. A redirected regulatory T lymphocyte (Treg cell) endowed withspecificity toward a selected target antigen or ligand, which cellcomprises a chimeric nucleic acid that encodes a chimeric receptorpolypeptide that comprises, expressed in a single, continuous chain, anextracellular recognition region, a transmembrane region and anintracellular signaling region, and is expressed in the redirected Tregcell so as to display the extracellular region on the cell surface,wherein (a) said extracellular recognition region of said chimericreceptor is specific for the selected target antigen or ligand; and (b)said intracellular signaling region comprises a combination of T-cellsignaling polypeptide moieties, which combination of moieties, uponbinding of the extracellular recognition region to the selected targetantigen or ligand, triggers activation of the redirected Treg cells tocause suppression of T-cell mediated immunity.
 2. A redirected Treg cellin accordance with claim 1, wherein said extracellular recognitionregion comprises an antibody-derived scFv domain that is specific for aselected antigen.
 3. A redirected Treg cell in accordance with claim 1,wherein said extracellular recognition region comprises a member of aligand-receptor pair, which is specific for the other member of thatpair.
 4. A redirected Treg cell in accordance with claim 1, wherein saidextracellular recognition region does not comprise an MHC proteinextracellular domain.
 5. A redirected Treg cell in accordance with claim1, wherein said extracellular recognition region is linked to saidtransmembrane region through a flexible spacer.
 6. A redirected Tregcell in accordance with claim 5, wherein said flexible spacer is a hingefrom a molecule of the immunoglobulin superfamily.
 7. A redirected Tregcell in accordance with claim 1, wherein said intracellular regioncomprises a signaling moiety from (i) a polypeptide chain of anantigen-specific receptor of a T-cell and/or (ii) a polypeptide chain ofa receptor of a T-cell that has a region comprising an immunoreceptortyrosine-based activation motif (ITAM).
 8. A redirected Treg cell inaccordance with claim 7, wherein said intracellular region comprises asignaling moiety from (ii).
 9. A redirected Treg cell in accordance withclaim 7, wherein said intracellular region comprises a signaling moietyfrom a polypeptide chain selected from the group consisting of a chainof the TCR/CD3 complex, and the γ chain of an Ig Fc receptor (FcRγ). 10.A redirected Treg cell in accordance with claim 9, wherein saidpolypeptide chain of an antigen-specific receptor of a T-cell is theCD3-ζ chain or the FcRγ chain.
 11. A redirected Treg cell in accordancewith claim 1, wherein said intracellular region includes a signalingmoiety of a T cell costimulatory-receptor protein.
 12. A redirected Tregcell in accordance with claim 11, wherein said costimulatory-receptorprotein is at least one protein selected from the group consisting ofCD28, OX40, CD40L, 4-1BB and PD-1.
 13. A redirected Treg cell inaccordance with claim 11, wherein said costimulatory-receptor protein isCD28.
 14. A redirected Treg cell in accordance with claim 11, whereinsaid costimulatory-receptor protein is 4-1BB.
 15. A redirected Treg cellin accordance with claim 1, wherein said intracellular region comprisesat least two different ones of said costimulatory-receptor proteinsignaling moieties.
 16. A redirected Treg cell in accordance with claim15, wherein said intracellular region comprises an intracellularsignaling moiety from CD28 and from 4-1BB.
 17. A redirected Treg cell inaccordance with claim 1, wherein said intracellular region comprises asignaling moiety from a T cell cytokine receptor.
 18. A redirected Tregcell in accordance with claim 17, wherein said cytokine receptor is theIL-2 receptor.
 19. A redirected Treg cell in accordance with claim 17,wherein said cytokine receptor is the TGF-β receptor.
 20. A redirectedTreg cell in accordance with claim 1, wherein said intracellularsignaling region includes a signal-transducing enzyme that (a) is anenzyme in the signal transduction pathway of an antigen-specificreceptor of a T-cell or (b) is an enzyme with corresponding specificityand activity as the enzyme of (a), derived from a non-T-cell lymphocyte.21. A redirected Treg cell in accordance with claim 20, wherein saidsignal-transducing enzyme is a member of the Syk-kinase family.
 22. Aredirected Treg cell in accordance with claim 1, wherein said chimericnucleic acid further includes a nucleotide sequence that will cause theredirected Treg to express Foxp3.
 23. A redirected Treg cell inaccordance with claim 1, wherein said selected target antigen or ligandis one that is present or expressed at a site or target tissue of anautoimmune or inflammatory response mediated by effector T-cells.
 24. Aredirected Treg cell in accordance with claim 23, wherein saidautoimmune or inflammatory response comprises an autoimmune response ordisease, an allograft or xenograft rejection, or graft-vs. host (GVH)disease.
 25. A redirected Treg cell in accordance with claim 1, whereinsaid target antigen or ligand is an autoantigen or an antigen that iscross-reactive with an autoantigen.
 26. A redirected Treg cell inaccordance with claim 25, wherein the autoantigen is a pathogenicantigen in the pathophysiology of said autoimmune disease.
 27. Theredirected Treg cell of claim 1, wherein said autoimmune or inflammatoryresponse and said target antigen or ligand are selected from the groupconsisting of: (a) inflammatory bowel disease (IBD), wherein saidantigen or ligand is one that is expressed in diseased colon or ileum;(b) rheumatoid arthritis, wherein said antigen or ligand is an epitopeof collagen or an antigen present in joints; (c) Type I diabetesmellitus or autoimmune insulitis, wherein said antigen or ligand is apancreatic β cell antigen; (d) multiple sclerosis, wherein said antigenor ligand is a myelin basic protein antigen, MOG-1, MOG-2 or anotherneuronal antigen; (e) autoimmune thyroiditis, wherein said antigen orligand is a thyroid antigen; (f) autoimmune gastritis, wherein saidantigen or ligand is a gastric antigen; (g) autoimmune uveitis oruveoretinitis, wherein said antigen or ligand is S-antigen or anotheruveal or retinal antigen; (h) autoimmune orchitis, wherein said antigenor ligand is a testicular antigen; (i)) autoimmune oophoritis, whereinsaid antigen or ligand is an ovarian antigen; (j) psoriasis; whereinsaid antigen or ligand is a keratinocyte antigen or another dermal orepidermal antigen; (k) vitiligo, where said antigen or ligand is amelanocyte antigen; (l) autoimmune prostatitis, wherein said antigen orligand is a prostate antigen; (m) any undesired immune response, whereinsaid antigen or ligand is an activation antigen expressed on T effectorcells present at the site of the undesired immune response; (n) tissuerejection, wherein said antigen or ligand is the MHC molecule having thehaplotype of the transplanted tissue, or a portion of that MHC molecule;and (o) an inflammatory condition, wherein said antigen or ligand is onethat is expressed on nonlymphoid cells of the hemopoietic lineage thatparticipate in inflammation.
 28. A redirected Treg cell in accordancewith claim 27, wherein said autoimmune disease is IBD and said antigenor ligand is carcinoembryonic antigen (CEA), or an antigen of intestinalbacterial flora.
 29. A redirected Treg cell in accordance with claim 27,wherein the activation antigen of (m) expressed on T effector cells isCD69 or CD107a.
 30. A redirected Treg cell in accordance with claim 27,wherein said target antigen or ligand of (o) is one that is expressed ona dendritic cell, macrophage/monocyte, granulocyte or eosinophil presentat said inflammation site.
 31. An immunoregulatory pharmaceuticalcomposition for suppressing a T effector cell-mediatedimmune/inflammatory response or treating a T effector cell-mediatedimmune/inflammatory disease or condition, comprising: (a) a Treg cellaccording to claim 1, and (b) a pharmaceutically and immunologicallyacceptable carrier, excipient or diluent.
 32. A method of producing theredirected Treg cell of claim 1, comprising: (a) obtaining from asubject and, optionally enriching or isolating and propagating, apopulation of lymphocytes or T-cells; (b) inducing a Treg cell phenotypein said cells by suitably stimulating or activating the cells byexposure to TGF-β or another cytokine that induces Foxp3 expressionthereby inducing the Treg phenotype; (c) before or after step (b),transducing the cells ex vivo with an expression vector encoding saidchimeric receptor to be expressed on said Treg cell; and (d) optionally,growing or expanding the cells obtained as above in vitro.
 33. A methodof producing the redirected Treg cell of claim 1, comprising: (a)obtaining from a subject and, optionally enriching or isolating andpropagating, a population of lymphocytes or T-cells; (b) transducing thecells ex vivo with a vector encoding the chimeric receptor; (c) before,after, or concomitantly with step (b), transducing the cells ex vivowith a recombinant nucleic acid expression construct encoding Foxp3; and(d) optionally, growing or expanding the cells obtained as above invitro.
 34. A method of suppressing undesired activity of T effectorcells in mediating an immune or inflammatory response, comprisingdelivering to a site of T effector cells to be suppressed an amount ofTreg cells according to claim 1 effective to suppress said T effectorcell activity.
 35. The method of claim 34 wherein said Treg cells areadministered systemically.
 36. The method of claim 34 wherein theredirected Treg cells are administered regionally or locally to a siteof inflammation.
 37. The method of claim 34 wherein said undesiredactivity of T effector cells is an autoimmune inflammatory response ordisorder, rejection of a transplant or GVH disease.
 38. A method fortreating or ameliorating symptoms of a disease or condition in amammalian subject that is mediated by undesired activity of T effectorcells, said method comprising administering to said subject in needthereof an effective amount of Treg cells in accordance with claim 1,wherein said target antigen or ligand is one that is present at the siteof said undesired T effector cell activity.
 39. The method of claim 38wherein the redirected Treg cells are produced by obtaining a populationof T-cells from the mammalian subject to be treated, transfecting saidcells with said chimeric nucleic acid and causing the cells to expressFoxp3 by (i) stimulating the transfected cells to induce Foxp3expression, or (ii) introducing an exogenous Foxp3-encoding constructinto the transfected cells.
 40. The method of claim 38 wherein theredirected Treg cells are produced by obtaining a mixed population ofT-cells from the mammalian subject to be treated, enriching or purifyingTreg cells from the mixed population of T-cells on the basis of the Tregcells' expression of CD4 and CD25 and/or Foxp3, and transfecting saidenriched or purified Treg cells with said chimeric nucleic acid.
 41. Themethod of claim 40 wherein the redirected Treg cells are produced by:(a) obtaining (i) peripheral blood mononuclear cells, (ii) peripheralblood lymphocytes, (iii) T-cells enriched or purified from (i) or (ii),or (iv) a subset of T-cells enriched or purified from (iii); (b)exposing the cells obtained in (a), ex vivo, to an amount of TGF-β orother Treg-inducing cytokine or agent that is effective to induceexpression of Foxp3 and convert T-cells to a Treg phenotype, (c)optionally, culturing and expanding said exposed cells of (a); and (d)before after or between said steps (a) and (b), transfecting said cellswith said chimeric nucleic acid.
 42. The method of claim 38 wherein saiddisease or condition comprises an autoimmune response or disease, anallograft or xenograft rejection, or graft-vs. host (GVH) disease. 43.The method of claim 38, wherein said disease or condition is IBD andsaid target antigen or ligand is CEA, or an antigen of intestinalbacterial flora.
 44. A method of claim 38, wherein said target antigenor ligand is LPS.
 45. A method of claim 44, wherein said extracellularrecognition region comprises an LPS-binding domain of MD-2 or anLPS-binding domain of CD-14 or both an LPS-binding domain of MD-2 and anLPS binding domain of CD-14.
 46. The method of claim 38, furtherincluding the step of, either before, concomitantly with, or afteradministration of the redirected Treg cells, introducing to a subject tothe site or target tissue of said immune or inflammatory response, anexogenous antigen or ligand and said target antigen or ligand is saidexogenous antigen or ligand.
 47. A chimeric DNA molecule comprising: (a)a first DNA segment comprising a sequence encoding an extracellularrecognition region specific for a selected target antigen or ligand,which region does not comprise an MHC protein extracellular domain, saidselected target antigen or ligand being one that is present or expressedat a site or tissue of a pathogenic or undesired immune responsemediated by effector T-cells; (b) a second DNA segment comprising asequence encoding a transmembrane region; and (c) a third DNA segmentcomprising a sequence encoding an intracellular signaling regioncomprising a combination of T-cell signaling polypeptide moieties, whichcombination of moieties trigger activation of the Treg cells to causesuppression of T-cell mediated immunity when the chimeric DNA istransfected into a Treg cell and said extracellular recognition regionbinds to the selected target antigen or ligand, wherein, upontransfection of the chimeric DNA into a Treg cell, the Treg cellexpresses said extracellular recognition region on its surface, saidtransmembrane region and said intracellular signaling region in onesingle, continuous chain.
 48. A chimeric receptor polypeptide encoded bythe chimeric DNA molecule of claim 47.